Decreasing expression level of proteasome subunit genes by treating a human subject with a nitroxide

ABSTRACT

Some embodiments disclosed herein include a method for decreasing gene expression of a gene. The methods can include identifying a human subject over the age of 35 and having an increase expression level of a gene associated with proteasome activity; and administering to the human subject an effective amount of a nitroxide antioxidant, whereby expression level of the gene is decreased.

BACKGROUND Field

The present disclosure relates generally to the field of modulation ofgene expression and more particularly to decreasing expression levels ofone or more genes relating to proteasome activity by treating humansubjects with a nitroxide.

Description of the Related Art

Genes generally encode biologically functional products. Gene expressiondescribes transcription of gene encoding DNA sequences intocomplementary DNA (cDNA) and translation of cDNA into the functionalproducts, such as proteins. Many factors, both internal and external,are involved in regulation of gene expression in cells. Such regulationcan manifest in an adjustment of gene expression to increase or decreasea number of proteins made.

Proteasomes (also referred to as proteasomes protein complexes) arepresent in cells. Many proteins act together to form and carryout thefunctions of the proteasome. These proteins that makeup the proteasomecomplex are examples of biologically functional products resulting fromgene expression.

Proteolysis is a process of degradation of proteins. Intracellularproteolysis is generally mediated by lysosome activity or proteasomeactivity. Proteasomes interact with, among other intracellularcomponents, ubiquitin tagged proteins marked for degradation, therebyinhibiting toxicity related to protein buildup within the cell.Proteasomes are present in eukaryotic cells at a high concentration andcleave peptides in an ATP/ubiquitin-dependent process in a non-lysosomalpathway

Certain conditions, such as cancer, can be associated with (e.g., causesor caused by) rapid and uncontrolled protein formation, and presence ofproteins that would otherwise be marked for degradation. In cellsassociated with the underlying condition, a toxic intracellularenvironment can be a desired method of treatment for the condition.Thus, inhibition or proteasome activity in such cells is essential fortreatment and prevention of certain conditions.

Other diseases and conditions can often be characterized by abnormalexpression of one or more genes. Irregularities in gene expressionunderlie many diseases and conditions. Overexpression or underexpressionof a gene or genes often results in dysfunction of downstream actionscontrolled by the same. Whether the gene is a regulator of cellularfunction or a vital in a responsive mechanism, modulation of the geneexpression is a fundamental directive in addressing the foundationalissues associated with many diseases and conditions.

SUMMARY

Some embodiments disclosed herein provide methods for decreasing geneexpression. The methods, in some embodiments, include identifying ahuman subject over the age of 35 and having an increase expression levelof a gene associated with proteasome activity; and administering to thehuman subject an effective amount of a nitroxide antioxidant resultingin a decreased expression level of the gene. In some embodiments, thegene is selected from the group consisting of: Psma6, Psmd3, Psmb4,Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3,Psmd13, Psma7, and Psmc4. In some embodiments, the human subject is overthe age of 45. In some embodiments, the human subject is over the age of55. In some embodiments, the human subject is over the age of 65. Insome embodiments, the expression level of the gene in a skin tissue isdecreased by treatment. In some embodiments, the expression level of thegene in an adipose tissue is decreased by treatment. In someembodiments, the expression level of the gene in blood is decreased bytreatment. In some embodiments, the expression level of the gene in aneuronal tissue is decreased by treatment. In some embodiments, thenitroxide antioxidant is 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl.In some embodiments, the effective amount of the nitroxide antioxidantis within a range of 0.01-300 mg/kg. In some embodiments, the effectiveamount of the nitroxide antioxidant is within a range of 0.1-250 mg/kg.In some embodiments, the effective amount of the nitroxide antioxidantis within a range of 1-200 mg/kg. In some embodiments, the effectiveamount of the nitroxide antioxidant is within a range of 2-150 mg/kg. Insome embodiments, the effective amount of the nitroxide antioxidant iswithin a range of 5-125 mg/kg. In some embodiments, the effective amountof the nitroxide antioxidant is within a range of 7-100 mg/kg. In someembodiments, the effective amount of the nitroxide antioxidant is withina range of 10-75 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 15-30 mg/kg.

Some embodiments disclosed herein provide methods for decreasing theexpression level of a gene in a human subject in need thereof,comprising: identifying a human subject having an increased expressionlevel of a gene associated with proteasome activity; administering tothe human subject an effective amount of a nitroxide antioxidant,whereby the expression level of the gene associated with proteasomeactivity is decreased. In some embodiments, the gene is selected fromthe group consisting of: Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4,Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, andPsmc4. In some embodiments, the increased expression level of the geneis age-related. In some embodiments, the human subject is over the ageof 35. In some embodiments, the human subject is over the age of 45. Insome embodiments, the human subject is over the age of 55. In someembodiments, the human subject is over the age of 65. In someembodiments, the increased expression level of the gene isdisease-related. In some embodiments, the disease is selected from thegroup consisting of cancer, rheumatoid/osteoid arthritis, systemic lupuserythematosus (SLE), inflammatory bowel disease, Alzheimer's disease,multiple sclerosis, atherosclerosis, cardiovascular disease, cataracts,dementia, osteoporosis, type 2 diabetes, and hypertension. In someembodiments, the disease is age-related. In some embodiments, theexpression level of the gene in a skin tissue is decreased by treatment.In some embodiments, the expression level of the gene in an adiposetissue is decreased by treatment. In some embodiments, the expressionlevel of the gene in blood is decreased by treatment. In someembodiments, the expression level of the gene in a neuronal tissue isdecreased by treatment. In some embodiments, the nitroxide antioxidantis 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl. In some embodiments,the effective amount of the nitroxide antioxidant is within a range of0.01-300 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 0.1-250 mg/kg. In someembodiments, the effective amount of the nitroxide antioxidant is withina range of 1-200 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 2-150 mg/kg. In someembodiments, the effective amount of the nitroxide antioxidant is withina range of 5-125 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 7-100 mg/kg. In someembodiments, the effective amount of the nitroxide antioxidant is withina range of 10-75 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 15-30 mg/kg.

Some embodiments disclosed herein provide methods for reducing risk of adisease in a human subject in need thereof, comprising: identifying ahuman subject over the age of 35 having an increased risk of a diseasedue to an increased expression level of a gene associated withproteasome activity; administering to the human subject an effectiveamount of a nitroxide antioxidant, whereby the expression level of thegene associated with proteasome activity is decreased. In someembodiments, the disease is selected from the group consisting ofcancer, rheumatoid/osteoid arthritis, systemic lupus erythematosus(SLE), inflammatory bowel disease, Alzheimer's disease, multiplesclerosis, atherosclerosis, cardiovascular disease, cataracts, dementia,osteoporosis, type 2 diabetes, and hypertension. In some embodiments,the gene is selected from the group consisting of: Psma6, Psmd3, Psmb4,Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3,Psmd13, Psma7, and Psmc4. In some embodiments, the human subject is overthe age of 45. In some embodiments, the human subject is over the age of55. In some embodiments, the human subject is over the age of 65. Insome embodiments, the expression level of the gene in a skin tissue isdecreased by treatment. In some embodiments, the expression level of thegene in an adipose tissue is decreased by treatment. In someembodiments, the expression level of the gene in blood is decreased bytreatment. In some embodiments, the expression level of the gene in aneuronal tissue is decreased by treatment. In some embodiments, thenitroxide antioxidant is 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl.In some embodiments, the effective amount of the nitroxide antioxidantis within a range of 0.01-300 mg/kg. In some embodiments, the effectiveamount of the nitroxide antioxidant is within a range of 0.1-250 mg/kg.In some embodiments, the effective amount of the nitroxide antioxidantis within a range of 1-200 mg/kg. In some embodiments, the effectiveamount of the nitroxide antioxidant is within a range of 2-150 mg/kg. Insome embodiments, the effective amount of the nitroxide antioxidant iswithin a range of 5-125 mg/kg. In some embodiments, the effective amountof the nitroxide antioxidant is within a range of 7-100 mg/kg. In someembodiments, the effective amount of the nitroxide antioxidant is withina range of 10-75 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 15-30 mg/kg.

Some embodiments disclosed herein provide methods comprising:identifying a human subject having or at risk of developing a cancer andin need of a decreased expression level of a gene associated withproteasome activity; administering to the human subject an effectiveamount of a nitroxide antioxidant, whereby the expression level of thegene associated with proteasome activity is decreased. In someembodiments, the cancer is associated with (e.g., correlates with) anincreased expression level of the gene associated with proteasomeactivity. The increased expression level of the gene associated withproteasome activity can be a response to rapid cell growth associatedwith the cancer. Rapid cell growth associated with the cancer mayrequire degradation of ubiquitin-tagged dysfunctional (e.g., improperlyfolded) proteins. In some embodiments, the cancer can be selected fromthe group consisting of bladder cancer, colorectal cancer,hepatocellular carcinoma, prostate carcinoma, and kidney carcinoma. Insome embodiments, the gene is selected from the group consisting of:Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4,Psma2, Psmc2, Psmc3, Psmd13, Psma7, and Psmc4. In some embodiments, thecancer is age-related. In some embodiments, the human subject is overthe age of 35. In some embodiments, the human subject is over the age of45. In some embodiments, the human subject is over the age of 55. Insome embodiments, the human subject is over the age of 65. In someembodiments, the expression level of the gene in a skin tissue isdecreased by treatment. In some embodiments, the expression level of thegene in an adipose tissue is decreased by treatment. In someembodiments, the expression level of the gene in blood is decreased bytreatment. In some embodiments, the expression level of the gene in aneuronal tissue is decreased by treatment. In some embodiments, thenitroxide antioxidant is 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl.In some embodiments, the effective amount of the nitroxide antioxidantis within a range of 0.01-300 mg/kg. In some embodiments, the effectiveamount of the nitroxide antioxidant is within a range of 0.1-250 mg/kg.In some embodiments, the effective amount of the nitroxide antioxidantis within a range of 1-200 mg/kg. In some embodiments, the effectiveamount of the nitroxide antioxidant is within a range of 2-150 mg/kg. Insome embodiments, the effective amount of the nitroxide antioxidant iswithin a range of 5-125 mg/kg. In some embodiments, the effective amountof the nitroxide antioxidant is within a range of 7-100 mg/kg. In someembodiments, the effective amount of the nitroxide antioxidant is withina range of 10-75 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 15-30 mg/kg.

Some embodiments disclosed herein provide methods comprising:identifying a human subject having or at risk of developing anautoimmune disease and in need of a decreased expression level of a geneassociated with proteasome activity; administering to the human subjectan effective amount of a nitroxide antioxidant, wherein the expressionlevel of the gene associated with proteasome activity is decreased. Insome embodiments, the autoimmune disease can be selected from the groupconsisting of rheumatoid/osteoid arthritis, systemic lupus erythematosus(SLE), inflammatory bowel disease, multiple sclerosis, atherosclerosis,and osteoporosis. In some embodiments, the gene is selected from thegroup consisting of: Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9,Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, and Psmc4. Insome embodiments, the gene is Cd51. In some embodiments, the autoimmunedisease is age-related. In some embodiments, the human subject is overthe age of 35. In some embodiments, the human subject is over the age of45. In some embodiments, the human subject is over the age of 55. Insome embodiments, the human subject is over the age of 65. In someembodiments, the expression level of the gene in a skin tissue isdecreased by treatment. In some embodiments, the expression level of thegene in an adipose tissue is decreased by treatment. In someembodiments, the expression level of the gene in blood is decreased bytreatment. In some embodiments, the expression level of the gene in aneuronal tissue is decreased by treatment. In some embodiments, thenitroxide antioxidant is 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl.In some embodiments, the effective amount of the nitroxide antioxidantis within a range of 0.01-300 mg/kg. In some embodiments, the effectiveamount of the nitroxide antioxidant is within a range of 0.1-250 mg/kg.In some embodiments, the effective amount of the nitroxide antioxidantis within a range of 1-200 mg/kg. In some embodiments, the effectiveamount of the nitroxide antioxidant is within a range of 2-150 mg/kg. Insome embodiments, the effective amount of the nitroxide antioxidant iswithin a range of 5-125 mg/kg. In some embodiments, the effective amountof the nitroxide antioxidant is within a range of 7-100 mg/kg. In someembodiments, the effective amount of the nitroxide antioxidant is withina range of 10-75 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 15-30 mg/kg.

Some embodiments disclosed herein provide methods for a diseaseassociated with an increased proteasome activity in a patient in needthereof, comprising: identifying a human subject having or at risk ofdeveloping a disease associated with an increased expression of a geneassociated with proteasome activity; administering to the human subjectan effective amount of a nitroxide antioxidant, whereby the expressionlevel of a gene associated with proteasome activity is decreased. Insome embodiments, the disease can be selected from the group consistingof cancer, rheumatoid/osteoid arthritis, systemic lupus erythematosus(SLE), inflammatory bowel disease, Alzheimer's disease, multiplesclerosis, atherosclerosis, cardiovascular disease, cataracts, dementia,osteoporosis, type 2 diabetes, and hypertension. In some embodiments,the gene is selected from the group consisting of: Psma6, Psmd3, Psmb4,Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3,Psmd13, Psma7, and Psmc4. In some embodiments, the human subject is overthe age of 35. In some embodiments, the human subject is over the age of45. In some embodiments, the human subject is over the age of 55. Insome embodiments, the human subject is over the age of 65. In someembodiments, the expression level of the gene in a skin tissue isdecreased by treatment. In some embodiments, the expression level of thegene in an adipose tissue is decreased by treatment. In someembodiments, the expression level of the gene in blood is decreased bytreatment. In some embodiments, the expression level of the gene in aneuronal tissue is decreased by treatment. In some embodiments, thenitroxide antioxidant is 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl.In some embodiments, the effective amount of the nitroxide antioxidantis within a range of 0.01-300 mg/kg. In some embodiments, the effectiveamount of the nitroxide antioxidant is within a range of 0.1-250 mg/kg.In some embodiments, the effective amount of the nitroxide antioxidantis within a range of 1-200 mg/kg. In some embodiments, the effectiveamount of the nitroxide antioxidant is within a range of 2-150 mg/kg. Insome embodiments, the effective amount of the nitroxide antioxidant iswithin a range of 5-125 mg/kg. In some embodiments, the effective amountof the nitroxide antioxidant is within a range of 7-100 mg/kg. In someembodiments, the effective amount of the nitroxide antioxidant is withina range of 10-75 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 15-30 mg/kg.

Some embodiments disclosed herein provide methods for treating anindividual in need thereof, comprising: identifying an individual overthe age of 35 in need of an increased expression level of a geneassociated with proteasome activity; and administering to the individualan effective amount of a nitroxide antioxidant to decrease the level ofexpression of the gene associated with proteasome activity. In someembodiments, the gene is selected from the group consisting of: Psma6,Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2,Psmc2, Psmc3, Psmd13, Psma7, and Psmc4. In some embodiments, the humansubject is over the age of 45. In some embodiments, the human subject isover the age of 55. In some embodiments, the human subject is over theage of 65. In some embodiments, the human subject has an increasedexpression level of the gene. In some embodiments, the individual has oris at risk of developing an age-related condition. In some embodiments,the age-related condition comprises increased senescence in a tissue. Insome embodiments, the age-related condition comprises hyperactivation ofproteasome activity in a tissue. In some embodiments, the age-relatedcondition comprises increased molecular heterogeneity. In someembodiments, the age-related condition comprises increased functionalimpairment in a tissue. In some embodiments, the expression level of thegene in a skin tissue is decreased by treatment. In some embodiments,the expression level of the gene in an adipose tissue is decreased bytreatment. In some embodiments, the expression level of the gene inblood is decreased by treatment. In some embodiments, the expressionlevel of the gene in a neuronal tissue is decreased by treatment. Insome embodiments, the nitroxide antioxidant is4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl. In some embodiments, theeffective amount of the nitroxide antioxidant is within a range of0.01-300 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 0.1-250 mg/kg. In someembodiments, the effective amount of the nitroxide antioxidant is withina range of 1-200 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 2-150 mg/kg. In someembodiments, the effective amount of the nitroxide antioxidant is withina range of 5-125 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 7-100 mg/kg. In someembodiments, the effective amount of the nitroxide antioxidant is withina range of 10-75 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 15-30 mg/kg.

Some embodiments disclosed herein provide methods for treating anindividual in need thereof, comprising: identifying an individual havinga disease-related increased expression level of a gene associated withproteasome activity; and administering to the individual an effectiveamount of a nitroxide antioxidant to decrease the level of expression ofthe gene associated with proteasome activity. In some embodiments, thedisease can be selected from the group consisting of cancer,rheumatoid/osteoid arthritis, systemic lupus erythematosus (SLE),inflammatory bowel disease, Alzheimer's disease, multiple sclerosis,atherosclerosis, cardiovascular disease, cataracts, dementia,osteoporosis, type 2 diabetes, and hypertension. In some embodiments,the gene is selected from the group consisting of: Psma6, Psmd3, Psmb4,Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3,Psmd13, Psma7, and Psmc4. In some embodiments, the human subject is overthe age of 35. In some embodiments, the human subject is over the age of45. In some embodiments, the human subject is over the age of 55. Insome embodiments, the human subject is over the age of 65. In someembodiments, the expression level of the gene in a skin tissue isdecreased by treatment. In some embodiments, the expression level of thegene in an adipose tissue is decreased by treatment. In someembodiments, the expression level of the gene in blood is decreased bytreatment. In some embodiments, the expression level of the gene in aneuronal tissue is decreased by treatment. In some embodiments, thenitroxide antioxidant is 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl.In some embodiments, the effective amount of the nitroxide antioxidantis within a range of 0.01-300 mg/kg. In some embodiments, the effectiveamount of the nitroxide antioxidant is within a range of 0.1-250 mg/kg.In some embodiments, the effective amount of the nitroxide antioxidantis within a range of 1-200 mg/kg. In some embodiments, the effectiveamount of the nitroxide antioxidant is within a range of 2-150 mg/kg. Insome embodiments, the effective amount of the nitroxide antioxidant iswithin a range of 5-125 mg/kg. In some embodiments, the effective amountof the nitroxide antioxidant is within a range of 7-100 mg/kg. In someembodiments, the effective amount of the nitroxide antioxidant is withina range of 10-75 mg/kg. In some embodiments, the effective amount of thenitroxide antioxidant is within a range of 15-30 mg/kg.

Some embodiments disclosed herein provide methods for treating anindividual having or at risk of developing a condition due to aging,comprising: identifying an individual over the age of 35; andadministering to the individual an effective amount of a nitroxideantioxidant, whereby the expression level of the gene associated withproteasome activity is decreased. In some embodiments, the individualhas an increased expression level of the gene. In some embodiments, thegene is selected from the group consisting of: Psma6, Psmd3, Psmb4,Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3,Psmd13, Psma7, and Psmc4. In some embodiments, the condition is anage-related condition. In some embodiments, the age-related conditioncomprises increased senescence in a tissue. In some embodiments, theage-related condition comprises overactivation of proteasome activity ina tissue. In some embodiments, the age-related condition comprisesincreased molecular heterogeneity. In some embodiments, the age-relatedcondition comprises increased functional impairment in a tissue. In someembodiments, the age-related condition is selected from the groupconsisting of cancer, rheumatoid/osteoid arthritis, systemic lupuserythematosus (SLE), inflammatory bowel disease, Alzheimer's disease,multiple sclerosis, atherosclerosis, cardiovascular disease, cataracts,dementia, osteoporosis, type 2 diabetes, and hypertension. In someembodiments, the human subject is over the age of 35. In someembodiments, the human subject is over the age of 45. In someembodiments, the human subject is over the age of 55. In someembodiments, the human subject is over the age of 65.

Some embodiments disclosed herein provide methods for decreasing theexpression level of a gene in a human subject in need thereof,comprising: identifying a human subject having an increased expressionlevel of a gene associated with proteasome activity, wherein the geneassociated with proteasome activity is selected from the groupconsisting of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12,Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, and Psmc4; anddelivering to the human subject an effective amount of a nitroxideantioxidant to reduce the level of expression of the gene associatedwith proteasome activity. In some embodiments, the nitroxide antioxidantis 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl. In some embodiments,the increased expression level of the gene is age-related. In someembodiments, wherein the increased expression level of the gene iscancer-related. In some embodiments, the increased expression level ofthe gene is disease-related. In some embodiments, the increasedexpression level of the gene is neurodegeneration-related. In someembodiments, the increased expression level of the gene is infectionrelated. In some embodiments, the reduced the level of expression of thegene initiates apoptosis. In some embodiments, the expression level ofthe gene is reduced in a tissue selected from the group consisting of askin tissue, an immune tissue, an adipose tissue, a pancreatic tissue,cardiac tissue, and a neuronal tissue by treatment.

Some embodiments disclosed herein provide methods for decreasing anexpression level, in an eukaryotic cell, of one or more genes encodingproteins involved in the proteasome complex by contacting the eukaryoticcell with a nitroxide antioxidant. In some embodiments, the one or moregenes is selected from the group consisting of Psma6, Psmd3, Psmb4,Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3,Psmd13, Psma7, and Psmc4. In some embodiments, the nitroxide antioxidantis 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl. In some embodiments,the eukaryotic cell is a cancer cell. In some embodiments, theexpression level of the one or more genes is decreased in said cell in atissue selected from the group consisting of a skin tissue, an immunetissue, an adipose tissue, a pancreatic tissue, cardiac tissue, and aneuronal tissue. In some embodiments, prior to said contacting, theeukaryotic cell exhibits an age-related increased expression level ofsaid one or more genes. In some embodiments, prior to said contacting,the eukaryotic cell exhibits a disease-related increased expressionlevel of said one or more genes. In some embodiments, prior to saidcontacting, the eukaryotic cell exhibits a neurodegeneration-relatedexpression level of said one or more genes.

Some embodiments disclosed herein provide methods for improvingchemotherapeutic response in a human subject comprising: contactingcancer cells in the subject with an effective amount of a nitroxideantioxidant whereby a level of expression of one or more genes encodingone or more proteasome subunits is decreased in said cancer cells. Insome embodiments, said cancer cells are known to have increasedproteasome activity. In some embodiments, the decreased expression levelof expression of one or more genes following treatment initiatesapoptosis within one or more of said cancer cells. In some embodiments,the nitroxide antioxidant is4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl. In some embodiments, theone or more genes is selected from the group consisting of Psma6, Psmd3,Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2,Psmc3, Psmd13, Psma7, and Psmc4.

Some embodiments disclosed herein provide methods for reducingproteasome activity in a human subject comprising: identifying a humansubject known to have increased proteasome activity; and delivering tothe subject an effective amount of a nitroxide antioxidant, whereby alevel of expression of a gene encoding one or more proteasome subunitsis decreased. In some embodiments, the nitroxide antioxidant is4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl. In some embodiments,increased proteasome activity is age-related. In some embodiments, theincreased proteasome activity is cancer-related. In some embodiments,the increased proteasome activity is disease-related. In someembodiments, the increased proteasome activity isneurodegeneration-related. In some embodiments, the increased proteasomeactivity is infection-related. In some embodiments, the decreased levelof expression of the gene initiates apoptosis. In some embodiments, theexpression level of the gene is decreased in a tissue selected from thegroup consisting of a skin tissue, an immune tissue, an adipose tissue,a pancreatic tissue, cardiac tissue, and a neuronal tissue followingtreatment.

Some embodiments disclosed herein provide methods for treating a humansubject having cancer comprising: delivering an effective amount of anitroxide antioxidant to a human subject, wherein the human subject haspreviously been administered at least one chemotherapeutic agent,whereby a level of expression of at least one gene encoding a proteasomeis decreased. In some embodiments, the human subject having cancer isidentified with an increased expression of one or more genes selectedfrom the group consisting of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4,Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, andPsmc4. In some embodiments, the methods further comprise administering aproteasome inhibitor to the human subject.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present disclosure belongs. See, e.g. Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley& Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y.1989). For purposes of the present disclosure, the following terms aredefined below.

All patents, applications, published applications and other publicationsreferred to herein are incorporated by reference for the referencedmaterial and in their entireties. If a term or phrase is used herein ina way that is contrary to or otherwise inconsistent with a definitionset forth in the patents, applications, published applications and otherpublications that are herein incorporated by reference, the use hereinprevails over the definition that is incorporated herein by reference.

As used herein, the term “expression” means the a gene product that isexpressed or produced by one or more nucleic acid molecules detectableby standard molecular biology methods, which gene product refers to e.g.an unspliced RNA, an mRNA, a splice variant mRNA, a polypeptide, apost-translationally modified polypeptide, a splice variant polypeptideetc., and specifically products made using an RNA gene product as atemplate, e.g. cDNA of the RNA.

As used herein, “differential expression” of a gene means that theexpression of the gene is at a higher level (“increased expression”) orlower level (“decreased expression”) in a human subject suffering from adisease, for example cancers and autoimmune diseases, relative to itsexpression in a normal or control subject. Differential expressionincludes both quantitative, as well as qualitative, differences in thetemporal or cellular expression pattern in a gene or its expressionproducts among, for example, normal and diseased cells, or among cellswhich have undergone different disease events or disease stages.

As used herein, “decreasing the expression level” of a gene meanscausing the expression of the gene to decrease by treating the humansubject with a compound, for example a nitroxide antioxidant, such thatthe expression level of the gene after treatment is lower than theexpression level of the gene before treatment in the human subject.

As used herein, “delivering” a compound means bringing that compoundinto contact with a relevant cell, tissue, or organism. Similarly,“contacting” means that the compound contacts a relevant target, such asa tissue or cell or tumor. In either case, delivery or contact in anorganism can be affected by directly administering the compound to theorganism, or by administering a different compound to the organism, suchas a prodrug that is converted in vivo to the desired compound. Inshort, these terms cover any action that leads to contact between thedesired compound and a target cell, tissue, or organism.

The present disclosure describes methods of modulating gene expressionin human subjects. However, this is illustrative only and not intendedto be limiting. For example, the methods disclosed herein can be usedfor modulating gene expression in other vertebrates, such as but notlimited to mammals, birds, reptiles, fish, and the like (withmodifications wherein appropriate). Mammals and birds include mostagricultural animals. Treatment of companion animals, e.g., dogs, cats,or birds is also contemplated.

It is understood that aspects and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

Other objects, advantages and features of the present invention willbecome apparent from the following specification taken in conjunctionwith the accompanying drawings.

Disclosed herein include methods for preventing, ameliorating, ortreating one or more conditions in a subject (e.g., a human subject) inneed thereof. The one or more conditions can be associated with (e.g.,caused by) a disease (e.g., cancer, autoimmune diseases, etc.). Theconditions may be associated with (e.g., correlated with) an increasedlevel (e.g., expression level) of proteasome genes. In some embodiments,a nitroxide antioxidant (e.g.,4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is administered to thesubject, whereby the expression level of one or more proteasome genes.Administering of the nitroxide antioxidant in the subject can result ina decreased level of one or more proteasome genes. The involvement of aproteasome gene in a particular condition may relate to the ability forthe ubiquitin mediated proteasome degradation of the ubiquitin-taggedproteins. For example, in cancer, cells rapidly produce large amounts ofdysfunctional proteins that require an increase in proteasome activityto accommodate the degradation of the dysfunctional proteins. Thisincrease may be a result of various mechanisms, including theuncontrolled proliferation of the cells. In some embodiments, anitroxide antioxidant can be, or act like, a proteasome inhibitor thatpromotes apoptosis. In some embodiments, decreased expression level ofproteasome genes prevents, ameliorates, or treats the one or moreconditions.

Human Subject Identification

The present disclosure relates to methods of treating alteration in geneexpression, such as age-related, cancer-related, disease-related,neurodegeneration-related, and infection-related alteration in geneexpression. Gene expression changes also play important roles in agingand serve as biomarkers of physiological decline and disease conditions,such as neurodegenerative diseases, and cancers. Therefore, one aspectof the present disclosure is methods of treating a human subject havingan age-related, cancer-related, disease-related, neurodegenerationrelated, and/or infection-related increase in gene expression levels,such as those genes associated with proteasome activity. In someembodiments, the human subject can be identified based on the humansubject's age, gene expression level, family history, health conditions,medical history, habits, or a combination thereof.

Regardless of the cause of the upregulation, some common terminology canbe used. In some embodiments, the expression level of a gene (e.g., agene associated with proteasome activity) in a human subject isconsidered to be upregulated or increased if the increase in theexpression level of that gene is statistically significant compared tothat of a control or a reference. In some embodiments, the expressionlevel of a gene (e.g., a gene associated with proteasome activity) in ahuman subject is considered to be upregulated or increased if theincrease in the expression level of that gene is statisticallysignificant compared to that of a control or a reference. The control orreference can be, for example, a normal healthy population, a populationat large, a collection of individuals of the same age or condition orsex, or the same human subject at a different time (e.g., at an earliertime of life when the human subject does or does not have the disease orcondition that results in the upregulation).

In some embodiments, a normal healthy population or a population atlarge can be a population having the same or similar gender, age, and/orrace, compared to the human subject. In some embodiments, the expressionlevel of the gene in the control or reference can be the mean or medianexpression level of the gene in control subjects in the control orreference subjects in the reference. The increase in expression levelcan be statistically significant if the probability of the observeddifference occurring not by chance, the confidence level, is greaterthan a threshold. The threshold can be, or be about, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or a number or a rangebetween any two of these values.

In some embodiments, the increase in expression level can be, or beabout, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, or a number or a range between any two of these values. In someembodiments, the increase in expression level can be at least 0.1%,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.

In some embodiments, the human subject may have an age that is, isabout, or is over 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100 years old.

In some embodiments, the human subject is identified based on the humansubject's expression profiles of one or more genes associated withproteasome activity. Non-limiting exemplary methods for determining thehuman subject's expression profiles include: amplification techniquessuch as PCR and RT-PCR (including quantitative variants), hybridizationtechniques such as in situ hybridization, microarrays, blots, andothers, and high throughput sequencing techniques like Next GenerationSequencing (Illumina, Roche Sequencer, Life Technologies SOLID™), SingleMolecule Real Time Sequencing (Pacific Biosciences), True SingleMolecule Sequencing (Helicos), or sequencing methods using no lightemitting technologies but other physical methods to detect thesequencing reaction or the sequencing product, like Ion Torrent (LifeTechnologies). Non-limiting exemplary methods for determining the humansubject's expression profiles include: binding techniques such as ELISA,immunohistochemistry, microarray and functional techniques such asenzymatic assays.

Genes Associated with Proteasome Activity

In some embodiments, administering to the human subject an effectiveamount of the nitroxide antioxidant results in an increased expressionlevel of a gene, for example a gene associated with proteasome activity.Therefore, some embodiments disclosed herein provide methods fortreating an individual in need thereof, comprising identifying anindividual having a disease-related increased expression level of a geneassociated with proteasome activity; and administering to the individualan effective amount of a nitroxide antioxidant to decrease the level ofexpression of the gene associated with proteasome activity. Someembodiments disclosed herein provide methods for treating an individualin need thereof, comprising identifying an individual in need of adecreased expression level of a gene associated with proteasomeactivity; and administering to the individual an effective amount of anitroxide antioxidant to decrease the level of expression of the geneassociated with proteasome activity. Some embodiments disclosed hereinprovide methods for treating an individual in need thereof, comprising:administering to the individual, known to have a disease-relatedincreased expression level of a gene associated with proteasomeactivity, an effective amount of a nitroxide antioxidant to decrease thelevel of expression of the gene associated with proteasome activity.Some embodiments disclosed herein provide methods for treating anindividual in need thereof, comprising: administering to an individual,known to be in need of a decreased expression level of a gene associatedwith proteasome activity, an effective amount of a nitroxide antioxidantto decrease the level of expression of the gene associated withproteasome activity.

Non-limiting examples of diseases associated with altered level ofproteasome activity include cancer; breast cancer; lung cancer; kidneycancer; cancers of the ovary and uterus; cancer of the central nervoussystem; cancers of the head and neck; melanoma; lymphomas; leukemia;neurological disorders; Alzheimer's disease; Parkinson's disease;Huntington's disease; amyotrophic lateral sclerosis; stroke;cardiovascular disorders; ischemia; heart failure; infections,infectious diseases; bacterial infections; inflammatory responses; viralinfections; autoimmune diseases; systemic lupus erythematosus;autoimmune lymphoproliferative syndrome; rheumatoid arthritis; andthyroiditis.

Non-limiting exemplary genes involved in proteasome activity includethose classified as Psma1, Psma2, Psma3, Psma4, Psma5, Psma6, Psma7,Psma8, Psme1, Psme2, Psme3, Psme4, Psmf, Psmg1, Psmg2, Psmg3, Psmg4,Shfm1, Pomp, Paaf1, Kiaa0368, Usp14, Hch15, Ube3C, Ube3A, Park2,Rad23A/B, Ubqln1/2; classified as beta-type subunits Psmb1, Psmb2,Psmb3, Psmb4, Psmb5, Psmb6, Psmb7, Psmb8, Psmb9, Psmb10, Psmb11;classified as ATPase subunits Psmc1, Psmc2, Psmc3, Psmc4, Psmc5, Psmc6;classified as non-ATPase subunits Psmd1, Psmd2, Psmd3, Psmd4, Psmd5,Psmd6, Psmd7, Psmd8, Psmd9, Psmd10, Psmd11, Psmd12, Psmd13, Psmd14,Adrm1.

The gene associated with proteasome activity can be Psma6, Psmd3, Psmb4,Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3,Psmd13, Psma7, or Psmc4. For example, the treatment can result indecreased expression levels of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4,Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, Psmc4,or any combination thereof. The decreased expression levels of Psma6,Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2,Psmc2, Psmc3, Psmd13, Psma7, Psmc4, or any combination thereof, candecreased the level of proteasome activity. The decreased level ofproteasome activity can result in a decrease in or disappearance ofsigns and symptoms of a disease associated with increased proteasomeactivity, including the curing of the disease associated with increasedproteasome activity. In some embodiments, the increased expressionlevels of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12,Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, Psmc4, or anycombination thereof, can decrease the level of proteasome activity. Thedecreased level of proteasome activity can result in a decrease in ordisappearance of signs and symptoms of the disease associated withincreased proteasome activity, including the curing of the diseaseassociated with increased proteasome activity. In some embodiments, thedecreased level of proteasome activity can inhibit, suppress, prevent,or reverse the disease or the symptoms associated with the disease.

Proteasome

The proteasome is a protein complex comprised of multiple subunitswithin eukaryotic cells. Functionally, proteasomes are configured todegrade unnecessary or dysfunctional proteins through proteolysis ofpeptide bonds within the proteins targeted for degradation. In this way,the proteasome regulates intracellular protein concentration generallypreventing cytotoxic environments known to trigger apoptosis.

The 26S proteasome, a multicatalytic protease complex over 2.5 MDa, isresponsible for degradation of a variety of proteins in cells, and thushelps maintain intracellular protein homeostasis (Ciechanover A.Proteolysis: from the lysosome to ubiquitin and the proteasome. Nat RevMol Cell Biol. 2005; 6:79-87; Finley D. Recognition and processing ofubiquitin-protein conjugates by the proteasome. Annu Rev Biochem. 2009;78:477-513; and Pickart C M, Cohen R E. Proteasomes and their kin:proteases in the machine age. Nat Rev Mol Cell Biol. 2004; 5:177-187;the content of each is incorporated herein by reference in itsentirety). In mammals, cytosolic proteasome 26S is generally comprisedof three main subunits. Two 19S cap subunits generally define terminalends of a 20S core subunit wherein proteolysis occurs. Both the cap andcore subunits are comprised of further subunits distinguishable by theircontribution to the overall proteasome complex. Alpha subunits aregenerally structural, while beta subunits within the 20S core arecatalytic to facilitate proteolysis.

The ubiquitin-proteasome system (UPS) is the major mechanism by whichproteins are degraded in the cytoplasm and nucleus of eukaryotic cellsand as such is a key player in maintaining protein homeostasis. Proteinsdestined to be degraded by the UPS are tagged for destruction byconjugation to the small protein ubiquitin through the action ofubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes, which canresult in the assembly of ubiquitin chains on one or more lysineresidues within the substrate. Proteins modified with an ubiquitin chainbind to ubiquitin receptors that link them to the 26S proteasome. The26S proteasome is a large proteolytic complex that degradesubiquitin-modified proteins and recycles the ubiquitin for future use.(Deshaies, Raymond J., Proteotoxic crisis, the ubiquitin-proteasomesystem, and cancer therapy. BMC Biology201412:94; the content of whichis incorporated herein by reference in its entirety).

The 20S proteasome is well characterized structurally (Tanaka, Keiji.“The Proteasome: Overview of Structure and Functions.” Proceedings ofthe Japan Academy. Series B, Physical and Biological Sciences85.1(2009): 12-36. PMC. Web. 29 Sept. 2017; the content of which isincorporated herein by reference in its entirety). It is awell-organized protein complex with a sedimentation coefficient of 20Sand a molecular mass of approximately 750 kDa. When viewed electronmicroscopically, the 20S proteasome appears as a cylinder-like structurein various eukaryotes, including yeast and mammals. It forms a packedparticle, a result of axial stacking of two outer β-rings and two innerα-rings, which are made up of seven structurally similar α and βsubunits, respectively; the rings form an α₁₋₇β₁₋₇β₁₋₇α₁₋₇ structure.The 20S proteasome plays essentially the same proteolytic roles in alleukaryotes, differing from proteasomes in prokaryotes that mainlyconsists of homo-hepatmeric α- and β-rings of the same α and β subunits,respectively, i.e., the αββα a structure. (Proteasomes from structure tofunction: perspectives from Archaea. Maupin-Furlow J A, Humbard M A,Kirkland P A, Li W, Reuter C J, Wright A J, Zhou G Curr Top Dev Biol.2006; 75( ):125-69.). The 20S proteasome processively degrades clientproteins, generating oligopeptides ranging in length from 3 to 15amino-acid residues. The resulting peptide products are subsequentlyhydrolyzed to amino acids by oligopeptidases and/or amino-carboxylpeptidases.

The enzymatically active proteasome is generally capped on either orboth ends of the central 20S proteasomal core by regulatory proteins(RP). The RP recognizes client proteins marked by polyubiquitin chains,removes the chain and entraps the protein moiety, unfolds the substrateproteins, opens the α-ring, and transfers the unfolded substrates intothe CP for destruction. The 19S RP comprises approximately 20 differentsubunits that can be subclassified into two groups: Regulatory particleof triple-ATPase (Rpt) subunits and Regulatory particle of non-ATPase(Rpn) subunits, both of which contain multiple proteins with molecularmasses ranging from 10 to 110 kDa. The following is a brief descriptionof the 19S RP, which comprises two sub-complexes: the lid and the base.(The regulatory particle of the Saccharomyces cerevisiae proteasome.Glickman M H, Rubin D M, Fried V A, Finley D Mol Cell Biol. 1998 Jun.18(6):3149-62; the content of which is incorporated by reference in itsentirety).

The catalytic core of the proteasome is a 20S cylinder, the inside ofwhich contains two copies each of the active sites β1, β2, and β5. Asecond form of the proteasome, referred to as the immunoproteasome, isenriched in cells of the hematopoietic lineage and has a specializedfunction in immune cells, but an essentially analogous composition inwhich the β1, β2, and β5 sites are replaced by the closely related β1i,β2i, and β5i sites. The β5/β5i sites (also known as thechymotrypsin-like sites) are inhibited by bortezomib with high potency,whereas the β1 (caspase-like) sites have approximately 10-fold loweraffinity and the β2 sites are not appreciably targeted under normalconditions. Substrates enter the 20S cylinder through its ends, whichare capped with structures referred to as 19S regulatory particles(RPs). A 20S cylinder capped at each end with a 19S RP is referred to asthe 26S proteasome. Assembly of the 26S proteasome is enabled by pocketsat the ends of the 20S cylinder into which are inserted shortcarboxy-terminal tails that emanate from a heterohexameric ring ofRpt1-6 subunits in the 19S RP. Degradation substrates are tethered tothe 26S proteasome via their ubiquitin chain, which binds to one or moreof a set of receptor proteins, some of which (for example, Rpn10 andRpn13) are intrinsic to the 19S RP, while others (for example, hRad23,hPLIC) shuttle on and off (Deshaies, Raymond J., Proteotoxic crisis, theubiquitin-proteasome system, and cancer therapy. BMC Biology201412:94;the content of which is incorporated by reference in its entirety).

Protein degradation by the proteasome can be ubiquitin dependent wherebya first ubiquitin binds to a protein targeted for degradation.Subsequently, more ubiquitin proteins bind to form a poly ubiquitinchain. Within the 19S cap, one or more ubiquitin receptors binds to thepolyubiquitin chain and directed the ubiquitin tagged protein within thecore of the proteasome for proteolysis.

In certain conditions, proteolytic activity and proteasome concentrationare increased when the cell attempts to reduce the concentration ofdysfunction, misfolded, or unnecessary proteins. For example, a cancercell requires increased proteasome activity to prevent a toxic buildupof dysfunctional proteins within the cell as a result of the unregulatedcellular function. Another example relates to oxidative stress wherebythe proteasome activates NF-kB by separating NF-kB from IkB in responseto TNFa or other external stress.

For example, cancer cells have a heightened dependence on mechanisms ofprotein homeostasis (proteostasis) including the UPS (Balch W E,Morimoto R I, Dillin A, Kelly J W: Adapting proteostasis for diseaseintervention. Science. 2008, 319: 916-919; the content of which isincorporated herein by reference in its entirety). Genome sequencing hasrevealed that cancer genomes are typically littered with dozens tohundreds of point mutations in protein coding sequences (Vogelstein B,Papadopoulos N, Velculescu V E, Zhou S, Diaz L A, Kinzler K W: Cancergenome landscapes. Science. 2013, 339: 1546-1558; the content of whichis incorporated herein by reference in its entirety). Many of thesemutated proteins are likely to present significant folding challenges,with increased degradation of the mutant protein via the UPS being onepossible outcome. In addition, cancer cell genomes often contain largeduplications, deletions, inversions, and translocations as well asaltered copy numbers of entire chromosomes (aneuploidy). It has beenestimated that over 90% of human solid tumors contain cells with morethan two copies of one or more chromosomes (Weaver B A, Cleveland D W:Does aneuploidy cause cancer?. Curr Opin Cell Biol. 2006, 18: 658-667;the content of which is incorporated herein by reference in itsentirety). These excess chromosomes continue to be expressed, andtherefore protein synthesis in aneuploid cancer cells is oftenimbalanced, with proteins encoded by extra chromosomes being produced inexcess over proteins encoded by chromosomes that are present in twocopies (Williams B R, Prabhu V R, Hunter K E, Glazier C M, Whittaker CA, Housman D E, Amon A: Aneuploidy affects proliferation and spontaneousimmortalization in mammalian cells. Science. 2008, 322: 703-709; thecontent of which is incorporated herein by reference in its entirety).This is particularly a problem for proteins that assemble to formstoichiometric complexes like the ribosome. In such cases, the excessproteins cannot attain stable conformations, and hence are degraded bythe UPS (Warner J R, Mitra G, Schwindinger W F, Studeny M, Fried H M:Saccharomyces cerevisiae coordinates accumulation of yeast ribosomalproteins by modulating mRNA splicing, translational initiation, andprotein turnover. Mol Cell Biol. 1985, 5: 1512-1521; the content ofwhich is incorporated herein by reference in its entirety). Thus, thismay create in cancer cells a heightened dependence on proteinquality-control (PQC) mechanisms, including protein chaperones, the UPS,and autophagy (Williams B R, Amon A: Aneuploidy: cancer's fatal flaw?.Cancer Res. 2009, 69: 5289-5291; the content of which is incorporatedherein by reference in its entirety).

The 26S proteasome is a multicatalytic proteinase complex with a highlyordered structure composed of 2 complexes, a 20S core and a 19Sregulator. The 20S core is composed of 4 rings of 28 non-identicalsubunits; 2 rings are composed of 7 alpha subunits and 2 rings arecomposed of 7 beta subunits. The 19S regulator is composed of a base,which contains 6 ATPase subunits and 2 non-ATPase subunits, and a lid,which contains up to 10 non-ATPase subunits. Proteasomes are distributedthroughout eukaryotic cells at a high concentration and cleave peptidesin an ATP/ubiquitin-dependent process in a non-lysosomal pathway. Anessential function of a modified proteasome, the immunoproteasome, isthe processing of class I MHC peptides.

Psma2

Psma2 is a gene that encodes the proteasome subunit alpha type-2, aprotein in humans which contributes to the complete assembly of the 20Sproteasome complex. As an alpha subunit, Psma2 structurally promotesinteraction between the 19s and 20s subunits. Such interaction isessential for general proteasome function. For example, the 19s subunit

Psma6

Proteasome subunit alpha type-6 is a protein that in humans is encodedby the Psma6 gene. This protein is one of the 17 essential subunits(alpha subunits 1-7, constitutive beta subunits 1-7, and induciblesubunits including betali, beta2i, beta5i) that contributes to thecomplete assembly of 20S proteasome complex.

Psma7

Proteasome subunit alpha type-7also known as 20S proteasome subunitalpha-4 is a protein that in humans is encoded by the PSMA7 gene Thisprotein is one of the 17 essential subunits (alpha subunits 1-7,constitutive beta subunits 1-7, and inducible subunits including beta1i,beta2i, beta5i) that contributes to the complete assembly of 20Sproteasome complex.

Psmb4

A Proteasome subunit beta type-4a, also known as 20S proteasome subunitbeta-7, is a protein that in humans is encoded by the PSMB4 gene. Thisprotein is one of the 17 essential subunits (alpha subunits 1-7,constitutive beta subunits 1-7, and inducible subunits including betali,beta2i, beta5i) that contributes to the complete assembly of 20Sproteasome complex. In particular, proteasome subunit beta type-2, alongwith other beta subunits, assembles into two heptameric rings andsubsequently a proteolytic chamber for substrate degradation. Theeukaryotic proteasome recognized degradable proteins, including damagedproteins for protein quality control purpose or key regulatory proteincomponents for dynamic biological processes. An essential function of amodified proteasome, the immunoproteasome, is the processing of class IMHC peptides.

Psmb5

A Proteasome subunit beta type-5, also known as 20S proteasome subunitbeta-5, is a protein that in humans is encoded by the Psmb5 gene. Thisprotein is one of the 17 essential subunits (alpha subunits 1-7,constitutive beta subunits 1-7, and inducible subunits including betali,beta2i, beta5i) that contributes to the complete assembly of 20Sproteasome complex. In particular, proteasome subunit beta type-5, alongwith other beta subunits, assembles into two heptameric rings andsubsequently a proteolytic chamber for substrate degradation. Thisprotein contains “chymotrypsin-like” activity and is capable of cleavingafter large hydrophobic residues of peptide. The eukaryotic proteasomerecognized degradable proteins, including damaged proteins for proteinquality control purpose or key regulatory protein components for dynamicbiological processes. An essential function of a modified proteasome,the immunoproteasome, is the processing of class I MHC peptides.

Psmc2

26S protease regulatory subunit 7, also known as 26S proteasomeAAA-ATPase subunit Rpt1, is an enzyme that in humans is encoded by thePSMC2 gene. This protein is one of the 19 essential subunits of acomplete assembled 19S proteasome complex. Six 26S proteasome AAA-ATPasesubunits (Rpt1 (this protein), Rpt2, Rpt3, Rpt4, Rpt5, and Rpt6)together with four non-ATPase subunits (Rpn1, Rpn2, Rpn10, and Rpn13)form the base sub-complex of 19S regulatory particle for proteasomecomplex.

Psmc3

26S protease regulatory subunit 6A, also known as 26S proteasomeAAA-ATPase subunit Rpt5, is an enzyme that in humans is encoded by thePsmc3 gene. This protein is one of the 19 essential subunits of acomplete assembled 19S proteasome complex. Six 26S proteasome AAA-ATPasesubunits (Rpt1, Rpt2, Rpt3, Rpt4, Rpt5 (i.e., Psmc3), and Rpt6) togetherwith four non-ATPase subunits (Rpn1, Rpn2, Rpn10, and Rpn13) form thebase sub complex of 19S regulatory particle for proteasome complex.

Psmc4

26S protease regulatory subunit 6B, also known as 26S proteasomeAAA-ATPase subunit Rpt3, is an enzyme that in humans is encoded by thePSMC4 gene. This protein is one of the 19 essential subunits of acomplete assembled 19S proteasome complex. Six 26S proteasome AAA-ATPasesubunits (Rpt1, Rpt2, Rpt3 (i.e., Psmc4), Rpt4, Rpt5, and Rpt6) togetherwith four non-ATPase subunits (Rpn1, Rpn2, Rpn10, and Rpn13) form thebase sub complex of 19S regulatory particle for proteasome complex.

Psmc6

26S protease regulatory subunit S10B, also known as 26S proteasomeAAA-ATPase subunit Rpt4, is an enzyme that in humans is encoded by thePsmc6 gene. This protein is one of the 19 essential subunits of acomplete assembled 19S proteasome complex. Six 26S proteasome AAA-ATPasesubunits (Rpt1, Rpt2, Rpt3, Rpt4 (Psmc6), Rpt5, and Rpt6) together withfour non-ATPase subunits (Rpn1, Rpn2, Rpn10, and Rpn13) form the basesub-complex of 19S regulatory particle for proteasome complex.

Psmd1

26S proteasome non-ATPase regulatory subunit 1, also as known as 26SProteasome Regulatory Subunit Rpn2, is a protein that in humans isencoded by the Psmd1 gene. This protein is one of the 19 essentialsubunits that contributes to the complete assembly of 19S proteasomecomplex.

Psmd3

26S proteasome non-ATPase regulatory subunit 3 is an enzyme that inhumans is encoded by the Psmd3 gene.

Psmd4

26S proteasome non-ATPase regulatory subunit 4, also as known as 26SProteasome Regulatory Subunit Rpn10(systematic nomenclature), is anenzyme that in humans is encoded by the PSMD4 gene. This protein is oneof the 19 essential subunits that contributes to the complete assemblyof 19S proteasome complex.

Psmd8

26S proteasome non-ATPase regulatory subunit 8 is an enzyme that inhumans is encoded by the Psmd8 gene. This gene encodes a non-ATPasesubunit of the 19S regulator. A pseudogene has been identified onchromosome 1.

Psmd9

26S proteasome non-ATPase regulatory subunit 9 is an enzyme that inhumans is encoded by the PSMD9 gene. This gene encodes a non-ATPasesubunit of the 19S regulator.

Psmd13

26S proteasome non-ATPase regulatory subunit 13 is an enzyme that inhumans is encoded by the PSMD13 gene.

Psmd14

26S proteasome non-ATPase regulatory subunit 14, also known as 26Sproteasome non-ATPase subunit Rpn11, is an enzyme that in humans isencoded by the Psmd14 gene. This protein is one of the 19 essentialsubunits of a complete assembled 19S proteasome complex. Nine subunitsRpn3, Rpn5, Rpn6, Rpn7, Rpn8, Rpn9, Rpn11, SEM1(Yeast analogue for humanprotein DSS1), and Rpn12 form the lid sub complex of 19S regulatoryparticle for proteasome complex.

Psme4

Proteasome activator complex subunit 4 is a protein that in humans isencoded by the PSME4 gene.

Methods for Counteracting or Treating a Condition (e.g., a Human SubjectOver a Certain Age) Aging

Some embodiments disclosed herein provide methods for counteractingincrease in gene expression (e.g., age-related increase or increase in ahuman subject that is over a certain age) or treating a disease (e.g.,an age-related disease or a disease in a human subject that is over acertain age), comprising (optionally) identifying a human subject overthe age of 35 and having an increased expression level of one or moregenes associated with proteasome activity or an age-related disease; andadministering to the human subject an effective amount of a nitroxideantioxidant. Healthy cells in a human subject over a certain age (e.g.,35 or 55) may have a natural decrease in proteasome activity. A diseaseor condition affecting an aging population may be associated withincreased proteasome activity. In some embodiments, the methods comprisedetermining the expression level of one or more genes associated withproteasome activity. The identification step and/or the determinationstep may not be necessary in some instances, such as where an increasedexpression level of one or more genes associated with proteasomeactivity can be inferred from the human subject's age, family history,health conditions, medical history, habits, or a combination thereof. Insome embodiments, the methods disclosed herein may be used to treat ahuman subject shows no symptoms of an age-related disease, but is atrisk of having an age-related disease. Exemplary risk factors for anage-related disease include, but are not limited to, age, familyhistory, health conditions, medical history, habits, or a combinationthereof. In some embodiments, risk factors for an age-related diseasecomprise an increased expression level of one or more genes associatedwith proteasome activity.

In some embodiments, administering to the human subject an effectiveamount of the nitroxide antioxidant results in a decreased expressionlevel of a gene, for example a gene associated with proteasome activity.The gene associated with proteasome activity can be Psma6, Psmd3, Psmb4,Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3,Psmd13, Psma7, or Psmc4. The treatment of the human subject with theeffective amount of the nitroxide antioxidant can result in a decreasedexpression level of the gene. For example, the treatment can result indecreased expression levels of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4,Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, Psmc4,or any combination thereof. The decreased expression levels of Psma6,Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2,Psmc2, Psmc3, Psmd13, Psma7, Psmc4, or any combination thereof, canincrease the level of apoptosis based on the resulting cytotoxic proteinbuildup within the cell associated with the decreased expression levels.The increased level of apoptosis can result in a decrease in ordisappearance of signs and symptoms of an age-related disease associatedwith increased proteasome activity, including the curing of theage-related disease. In some embodiments, the decreased expressionlevels of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12,Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, Psmc4, or anycombination thereof, can increase the level of apoptosis. The increasedlevel of apoptosis can result in a decrease in or disappearance of signsand symptoms of the age-related disease associated with increasedproteasome activity, including the curing of the disease associated withage-related disease associated with increased proteasome activity.

In some embodiments, the levels of Psma6, Psmd3, Psmb4, Psmb5, Psmc6,Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7,Psmc4, or any combination thereof in the connective tissue, muscletissue, nervous tissue, and/or epithelial tissue may change after thenitroxide antioxidant is administered. Non-limiting examples of theconnective tissue include dense connective tissue, loose connectivetissue, reticular connective tissue, adipose tissue, cartilage, bone,and extracellular matrix. Non-limiting examples of the muscle tissueincludes smooth muscle tissue, cardiac muscle tissue, and skeletalmuscle tissue. Non-limiting examples of the nervous tissue includeneural tissue of the central nervous system, neural tissue of theperipheral nervous system, the brain, spinal cord, cranial nerves,spinal nerves, and motor neurons. Non-limiting examples of theepithelial tissue include squamous epithelium, cuboidal epithelium,columnar epithelium, glandular epithelium, ciliated epithelium, andskin.

Some embodiments disclosed herein provide methods for treating a diseaserelated to aging in a human subject in need thereof, comprising(optionally) identifying a human subject over the age of 35 and havingan age-related disease and having an increased expression level of agene associated with proteasome activity; and administering to the humansubject an effective amount of a nitroxide antioxidant. Some embodimentsdisclosed herein provide methods for treating an individual having or atrisk of developing a condition due to aging, comprising: identifying anindividual over the age of 35; and administering to the individual aneffective amount of a nitroxide antioxidant, whereby the expressionlevel of the gene associated with proteasome activity is decreased.

Non-limiting examples of age-related diseases include cancer,rheumatoid/osteoid arthritis, systemic lupus erythematosus (SLE),inflammatory bowel disease, Alzheimer's disease, multiple sclerosis,atherosclerosis, cardiovascular disease, cataracts, dementia,osteoporosis, type 2 diabetes, hypertension.

Methods for Decreasing Expression Level of a Gene

Some embodiments disclosed herein provide methods for decreasing theexpression level of a gene in a human subject in need thereof,comprising (optionally) identifying a human subject having an increasedexpression level of a gene associated with proteasome activity; andadministering to the human subject an effective amount of a nitroxideantioxidant. Some embodiments disclosed herein provide methods fortreating a disease associated with an increased proteasome activity in apatient in need thereof, comprising (optionally) identifying a humansubject having an increased expression level of a gene associated withproteasome activity; and administering to the human subject an effectiveamount of a nitroxide antioxidant. The increased expression level may beage-related, or disease related. In some embodiments, the disease may becancer, rheumatoid/osteoid arthritis, systemic lupus erythematosus(SLE), inflammatory bowel disease, Alzheimer's disease, multiplesclerosis, atherosclerosis, cardiovascular disease, cataracts, dementia,osteoporosis, type 2 diabetes, hypertension, or any combination thereof.Some embodiments disclosed herein provide methods for treating anindividual in need thereof, comprising (optionally) identifying a humansubject over the age of 35 in need of a decreased expression level of agene associated with proteasome activity; and administering to the humansubject an effective amount of a nitroxide antioxidant. In someembodiments, the methods comprise determining the expression level ofone or more genes associated with proteasome activity. In someembodiments, the determination step comprises inferring increasedexpression level of one or more genes associated with proteasomeactivity based on the human subject's age, family history, healthconditions, medical history, habits, or a combination thereof. In someembodiments, the methods disclosed herein may be used to treat a humansubject shows no symptoms of a disease associated with increasedproteasome activity, but is at risk of having a disease associated withincreased proteasome activity. Exemplary risk factors for a diseaseassociated with increased proteasome activity include, but are notlimited to, age, family history, health conditions, medical history,habits, or a combination thereof.

In some embodiments, administering to the human subject an effectiveamount of the nitroxide antioxidant results in a decreased expressionlevel of a gene, for example a gene associated with proteasome activity.The gene associated with proteasome activity can be Psma6, Psmd3, Psmb4,Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3,Psmd13, Psma7, or Psmc4. The treatment of the human subject with theeffective amount of the nitroxide antioxidant can result in a decreasedexpression level of the gene. For example, the treatment can decreasethe expression levels of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4,Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, Psmc4,or any combination thereof. The decreased expression of the gene cancounteract the increase in the expression level of the gene.

Methods for Treating Cancer

Some embodiments disclosed herein provide methods for treating cancer ina human subject in need thereof, comprising (optionally) identifying ahuman subject having a cancer and in need of a decreased expressionlevel of a gene associated with proteasome activity; and administeringto the human subject an effective amount of a nitroxide antioxidant. Insome embodiments, the methods disclosed herein may be used to treat ahuman subject that shows no symptoms of cancer, but is at risk of havingcancer. Exemplary risk factors for cancer include, but are not limitedto, age, family history, health conditions, medical history, habits, ora combination thereof. In some embodiments, risk factors for cancercomprise an increased expression level of one or more genes associatedwith proteasome activity. Anti-apoptotic upregulation of the proteasomecomplex (or a gene of the proteasome complex) and associated activityhave been observed in cancer cells and accommodate increased productionof misfolded proteins (Arlt, et al. Increased proteasome subunit proteinexpression and proteasome activity in colon cancer relate to an enhancedactivation of nuclear factor E2-related factor 2 (Nrf2). Oncogene (2009)28:3983-3996; Almond, et al. The proteasome: a novel target for cancerchemotherapy. Leukemia (2002) 16:433-443; Chen et al, IncreasedProteasome Activity, Ubiquitin-Conjugating Enzymes, and eEF1ATranslation Factor Detected in Breast Cancer Tissue. Molecular Biology,Pathobiology and Genetics. Cancer Research (2005) 65(13):5599-5606; thecontent of each is incorporated herein by reference in its entirety).

Non-limiting examples of the methods for identifying a human subjecthaving a cancer include colonoscopy; sigmoidoscopy; and high-sensitivityfecal occult blood tests. In some embodiments, methods for identifying ahuman subject having a cancer include low-dose helical computedtomography; mammography; and pap test and human papillomavirus (HPV)testing. In some embodiments, methods for identifying a human subjecthaving a cancer include alpha-fetoprotein blood test; breast magneticresonance imaging (MRI); CA-125 test; clinical breast exams and regularbreast self-exams; prostate-specific antigen (PSA) testing; skin exams;transvaginal ultrasound; and virtual colonoscopy. In some embodiments,methods for identifying a human subject having a cancer include bariumenema; biopsy; bone marrow aspiration and biopsy; bone scan; breast MRIfor early detection of breast cancer; breast MRI; colonoscopy; computedtomography (CT) scan; digital rectal exam (DRE); blood and plateletstesting; bone marrow testing; umbilical cord blood testing;electrocardiogram (EKG) and echocardiogram; endoscopic techniques; fecaloccult blood tests; magnetic resonance imaging (MRI); mammography; multigated acquisition (MUGA) scan; papanicolaou (pap) test; positronemission tomography and computed tomography (PET-CT) scan;sigmoidoscopy; tumor marker tests; ultrasound; upper endoscopy. In someembodiments, methods for identifying a human subject having a cancerinclude DNA sequencing; detecting presence of single nucleotidepolymorphism (SNIP); and detecting the presence of certain proteinmarkers.

In some embodiments, administering to the human subject an effectiveamount of the nitroxide antioxidant results in a decreased expressionlevel of a gene, for example a gene associated with proteasome activity.The gene associated with proteasome activity can be Psma6, Psmd3, Psmb4,Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3,Psmd13, Psma7, or Psmc4. The treatment of the human subject with theeffective amount of the nitroxide antioxidant can result in a decreasedexpression of the gene. For example, the treatment can result indecreased expression levels of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4,Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, Psmc4,or any combination thereof. The decreased expression level of the genecan initiate apoptotic cascades resulting in programmed cell death. Theincreased level of apoptosis can result in a decrease in ordisappearance of signs and symptoms of the cancer, including the curingof the cancer.

Non-limiting examples of cancer include bladder and other urothelialcancers; breast cancer; cervical cancer; colorectal cancer; endometrialcancer; endometrial cancer; esophageal cancer; liver (hepatocellular)cancer; lung cancer; neuroblastoma cancer; oral cavity and oropharyngealcancer; ovarian, fallopian tube, and primary peritoneal cancer; prostatecancer; skin cancer; stomach (gastric) cancer; and testicular cancer.

Non-limiting examples of cancer include acute lymphoblastic leukemia,adult; acute myeloid leukemia, adult; adrenocortical carcinoma;aids-related lymphoma; anal cancer; bile duct cancer; bladder cancer;brain tumors, adult; breast cancer; breast cancer and pregnancy; breastcancer, male; carcinoid tumors, gastrointestinal; carcinoma of unknownprimary; cervical cancer; chronic lymphocytic leukemia; chronicmyelogenous leukemia; chronic myeloproliferative neoplasms; cnslymphoma, primary; colon cancer; endometrial cancer; esophageal cancer;extragonadal germ cell tumors; fallopian tube cancer; gallbladdercancer; gastric cancer; gastrointestinal carcinoid tumors;gastrointestinal stromal tumors; germ cell tumors, extragonadal; germcell tumors, ovarian; gestational trophoblastic disease; hairy cellleukemia; hepatocellular (liver) cancer, adult primary; histiocytosis,langerhans cell; hodgkin lymphoma, adult; hypopharyngeal cancer;intraocular (eye) melanoma; islet cell tumors, pancreatic neuroendocrinetumors; kaposi sarcoma; kidney (renal cell) cancer; kidney (renal pelvisand ureter, transitional cell) cancer; langerhans cell histiocytosis;laryngeal cancer; leukemia, adult acute lymphoblastic; leukemia, adultacute myeloid; leukemia, chronic lymphocytic; leukemia, chronicmyelogenous; leukemia, hairy cell; lip and oral cavity cancer; livercancer, adult primary; lung cancer, non-small cell; lung cancer, smallcell; lymphoma, adult Hodgkin; lymphoma, adult non-hodgkin; lymphoma,aids-related; lymphoma, primary cns; malignant mesothelioma; melanoma;melanoma, intraocular (eye); merkel cell carcinoma; metastatic squamousneck cancer with occult primary; multiple myeloma and other plasma cellneoplasms; mycosis fungoides and the sézary syndrome; myelodysplasticsyndromes; myelodysplastic/myeloproliferative neoplasms;myeloproliferative neoplasms, chronic; paranasal sinus and nasal cavitycancer; nasopharyngeal cancer; neck cancer with occult primary,metastatic squamous; non-hodgkin lymphoma, adult; non-small cell lungcancer; oral cavity cancer, lip oropharyngeal cancer; ovarian epithelialcancer; ovarian germ cell tumors; ovarian low malignant potentialtumors; pancreatic cancer; pancreatic neuroendocrine tumors (islet celltumors); pheochromocytoma and paraganglioma; paranasal sinus and nasalcavity cancer; parathyroid cancer; penile cancer; pheochromocytoma andparaganglioma; pituitary tumors; plasma cell neoplasms, multiple myelomaand other; breast cancer and pregnancy; primary peritoneal cancer;prostate cancer; rectal cancer; renal cell cancer; transitional cellrenal pelvis and ureter; salivary gland cancer; sarcoma, Kaposi;sarcoma, soft tissue, adult; sarcoma, uterine; mycosis fungoides and thesézary syndrome; skin cancer, melanoma; skin cancer, nonmelanoma; smallcell lung cancer; small intestine cancer; stomach (gastric) cancer;testicular cancer; thymoma and thymic carcinoma; thyroid cancer;transitional cell cancer of the renal pelvis and ureter; trophoblasticdisease, gestational; carcinoma of unknown primary; urethral cancer;uterine cancer, endometrial; uterine sarcoma; vaginal cancer; and vulvarcancer.

In some embodiments, non-limiting examples of cancer include, but arenot limited to, hematologic and solid tumor types such as acousticneuroma, acute leukemia, acute lymphoblastic leukemia, acute myelogenousleukemia (monocytic, myeloblastic, adenocarcinoma, angiosarcoma,astrocytoma, myelomonocytic and promyelocytic), acute t-cell leukemia,basal cell carcinoma, bile duct carcinoma, bladder cancer, brain cancer,breast cancer (including estrogen-receptor positive breast cancer),bronchogenic carcinoma, Burkitt's lymphoma, cervical cancer,chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chroniclymphocytic leukemia, chronic myelocytic (granulocytic) leukemia,chronic myelogenous leukemia, colon cancer, colorectal cancer,craniopharyngioma, cystadenocarcinoma, dysproliferative changes(dysplasias and metaplasias), embryonal carcinoma, endometrial cancer,endotheliosarcoma, ependymoma, epithelial carcinoma, erythroleukemia,esophageal cancer, estrogen-receptor positive breast cancer, essentialthrombocythemia, Ewing's tumor, fibrosarcoma, gastric carcinoma, germcell testicular cancer, gestational trophobalstic disease, glioblastoma,head and neck cancer, heavy chain disease, hemangioblastoma, hepatoma,hepatocellular cancer, hormone insensitive prostate cancer,leiomyosarcoma, liposarcoma, lung cancer (including small cell lungcancer and non-small cell lung cancer), lymphangioendothelio-sarcoma,lymphangiosarcoma, lymphoblastic leukemia, lymphoma (lymphoma, includingdiffuse large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphomaand non-Hodgkin's lymphoma), malignancies and hyPerproliferativedisorders of the bladder, breast, colon, lung, ovaries, pancreas,prostate, skin and uterus, lymphoid malignancies of T-cell or B-cellorigin, leukemia, medullary carcinoma, medulloblastoma, melanoma,meningioma, mesothelioma, multiple myeloma, myelogenous leukemia,myeloma, myxosarcoma, neuroblastoma, oligodendroglioma, oral cancer,osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillaryadenocarcinomas, papillary carcinoma, peripheral T-cell lymphoma,pinealoma, polycythemia vera, prostate cancer (includinghormone-insensitive (refractory) prostate cancer), rectal cancer, renalcell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceousgland carcinoma, seminoma, skin cancer, small cell lung carcinoma, solidtumors (carcinomas and sarcomas), stomach cancer, squamous cellcarcinoma, synovioma, sweat gland carcinoma, testicular cancer(including germ cell testicular cancer), thyroid cancer, Waldenstrom'smacroglobulinemia, testicular tumors, uterine cancer, Wilms' tumor andthe like.

Non-limiting examples of the cancer include acute lymphoblasticleukemia, childhood; acute myeloid leukemia/other myeloid malignancies,childhood; adrenocortical carcinoma, childhood; astrocytomas, childhood;atypical teratoid/rhabdoid tumor, childhood central nervous system;basal cell carcinoma, childhood; bladder cancer, childhood; bone,malignant fibrous histiocytoma of and osteosarcoma; brain and spinalcord tumors overview, childhood; brain stem glioma, childhood; (braintumor), childhood astrocytomas; (brain tumor), childhood central nervoussystem atypical teratoid/rhabdoid tumor; (brain tumor), childhoodcentral nervous system embryonal tumors; (brain tumor), childhoodcentral nervous system germ cell tumors; (brain tumor), childhoodcraniopharyngioma; (brain tumor), childhood ependymoma; breast cancer,childhood; bronchial tumors, childhood; carcinoid tumors, childhood;carcinoma of unknown primary, childhood; cardiac (heart) tumors,childhood; central nervous system atypical teratoid/rhabdoid tumor,childhood; central nervous system embryonal tumors, childhood; centralnervous system germ cell tumors, childhood; cervical cancer, childhood;chordoma, childhood; colorectal cancer, childhood; craniopharyngioma,childhood; effects, treatment for childhood cancer, late; embryonaltumors, central nervous system, childhood; ependymoma, childhood;esophageal tumors, childhood; esthesioneuroblastoma, childhood; ewingsarcoma; extracranial germ cell tumors, childhood; gastric (stomach)cancer, childhood; gastrointestinal stromal tumors, childhood; germ celltumors, childhood central nervous system; germ cell tumors, childhoodextracranial; glioma, childhood brain stem; head and neck cancer,childhood; heart tumors, childhood; hematopoietic cell transplantation,childhood; histiocytoma of bone, malignant fibrous and osteosarcoma;histiocytosis, langerhans cell; hodgkin lymphoma, childhood; kidneytumors of childhood, wilms tumor and other; langerhans cellhistiocytosis; laryngeal cancer, childhood; late effects of treatmentfor childhood cancer; leukemia, childhood acute lymphoblastic; leukemia,childhood acute myeloid/other childhood myeloid malignancies; livercancer, childhood; lung cancer, childhood; lymphoma, childhood Hodgkin;lymphoma, childhood non-Hodgkin; malignant fibrous histiocytoma of boneand osteosarcoma; melanoma, childhood; mesothelioma, childhood; midlinetract carcinoma, childhood; multiple endocrine neoplasia, childhood;myeloid leukemia, childhood acute/other childhood myeloid malignancies;nasopharyngeal cancer, childhood; neuroblastoma, childhood; non-hodgkinlymphoma, childhood; oral cancer, childhood; osteosarcoma and malignantfibrous histiocytoma of bone; ovarian cancer, childhood; pancreaticcancer, childhood; papillomatosis, childhood; paraganglioma, childhood;pediatric supportive care; pheochromocytoma, childhood; pleuropulmonaryblastoma, childhood; retinoblastoma; rhabdomyosarcoma, childhood;salivary gland cancer, childhood; sarcoma, childhood soft tissue;(sarcoma), ewing sarcoma; (sarcoma), osteosarcoma and malignant fibroushistiocytoma of bone; (sarcoma), childhood rhabdomyosarcoma; (sarcoma)childhood vascular tumors; skin cancer, childhood; spinal cord tumorsoverview, childhood brain and; squamous cell carcinoma (skin cancer),childhood; stomach (gastric) cancer, childhood; supportive care,pediatric; testicular cancer, childhood; thymoma and thymic carcinoma,childhood; thyroid tumors, childhood; transplantation, childhoodhematopoietic; childhood carcinoma of unknown primary; unusual cancersof childhood; vaginal cancer, childhood; vascular tumors, childhood; andwilms tumor and other childhood kidney tumors.

Non-limiting examples of cancer include embryonal rhabdomyosarcoma,pediatric acute lymphoblastic leukemia, pediatric acute myelogenousleukemia, pediatric alveolar rhabdomyosarcoma, pediatric anaplasticependymoma, pediatric anaplastic large cell lymphoma, pediatricanaplastic medulloblastoma, pediatric atypical teratoid/rhabdoid tumorof the central nervous system, pediatric biphenotypic acute leukemia,pediatric Burkitts lymphoma, pediatric cancers of Ewing's family oftumors such as primitive neuroectodermal rumors, pediatric diffuseanaplastic Wilm's tumor, pediatric favorable histology Wilm's tumor,pediatric glioblastoma, pediatric medulloblastoma, pediatricneuroblastoma, pediatric neuroblastoma-derived myelocytomatosis,pediatric pre-B-cell cancers (such as leukemia), pediatric psteosarcoma,pediatric rhabdoid kidney tumor, pediatric rhabdomyosarcoma, andpediatric T-cell cancers such as lymphoma and skin cancer.

Methods for Treating Autoimmune Diseases

Some embodiments disclosed herein provide methods for treating anautoimmune disease in a human subject in need thereof, comprising(optionally) identifying a human subject having an autoimmune diseaseand in need of a decreased expression level of a gene associated withproteasome activity; and administering to the human subject an effectiveamount of a nitroxide antioxidant. In some embodiments, the nitroxideantioxidant can be, or act like, a proteasome inhibitor. A proteasomeinhibitor can be a therapeutics of an autoimmune disease, such asrheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndromeand sclerodema (Verbrugge et al. Proteasome inhibitors as experimentaltherapeutics of autoimmune diseases. Arthritis Res Ther. 2015;17(1):1-10; the content of which is incorporated herein by reference inits entirety). In some embodiments, the methods disclosed herein may beused to treat a human subject shows no symptoms of an autoimmunedisease, but is at risk of having an autoimmune disease. Exemplary riskfactors for an autoimmune disease include, but are not limited to, age,family history, health conditions, medical history, habits, or acombination thereof. In some embodiments, risk factors for an autoimmunedisease comprise an increased expression level of one or more genesassociated with proteasome activity.

In some embodiments, Autoimmunity is the system of immune responses ofan organism against its own healthy cells and tissues. Any disease thatresults from such an aberrant immune response is termed an “autoimmunedisease”. Prominent examples include celiac disease, diabetes mellitustype 1, sarcoidosis, systemic lupus erythematosus (SLE), Sjögren'ssyndrome, eosinophilic granulomatosis with polyangiitis, Hashimoto'sthyroiditis, Graves' disease, idiopathic thrombocytopenic purpura,Addison's disease, rheumatoid arthritis (RA), ankylosing spondylitis,polymyositis (PM), and dermatomyositis (DM). Autoimmune diseases arevery often treated with steroids

In some embodiments, administering to the human subject an effectiveamount of the nitroxide antioxidant results in a decreased expressionlevel of a gene, for example a gene associated with proteasome activity.The gene associated with proteasome activity can be Psma6, Psmd3, Psmb4,Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3,Psmd13, Psma7, or Psmc4. The treatment of the human subject with theeffective amount of the nitroxide antioxidant can result in a decreasedexpression level of the gene. For example, the treatment can result indecreased expression levels of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4,Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, Psmc4,or any combination thereof. The increased expression levels of Psma6,Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2,Psmc2, Psmc3, Psmd13, Psma7, Psmc4, or any combination thereof, canincrease the level of apoptosis. The increased level of apoptosis canresult in a decrease in or disappearance of signs and symptoms of theautoimmune disease, including the curing of the autoimmune disease. Insome embodiments, the decreased expression levels of Psma6, Psmd3,Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2,Psmc3, Psmd13, Psma7, Psmc4, or any combination thereof, can increasethe level of apoptosis. The increased level of apoptosis can result in adecrease in or disappearance of signs and symptoms of the autoimmunedisease, including the curing of the autoimmune disease.

Non-limiting examples of autoimmune diseases include rheumatoidarthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis,Lyme arthritis, psoriatic arthritis, reactive arthritis,spondyloarthropathy, systemic lupus erythematosus, Crohn's disease,ulcerative colitis, inflammatory bowel disease, insulin dependentdiabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis,dermatitis scleroderma, graft versus host disease, organ transplantrejection, acute or chronic immune disease associated with organtransplantation, sarcoidosis, atherosclerosis, disseminatedintravascular coagulation, Kawasaki's disease, Grave's disease,nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis,Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys,chronic active hepatitis, uveitis, septic shock, toxic shock syndrome,sepsis syndrome, cachexia, infectious diseases, parasitic diseases,acquired immunodeficiency syndrome, acute transverse myelitis,Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke,primary biliary cirrhosis, hemolytic anemia, malignancies, heartfailure, myocardial infarction, Addison's disease, sporadic,polyglandular deficiency type I and polyglandular deficiency type II,Schmidt's syndrome, adult (acute) respiratory distress syndrome,alopecia, alopecia greata, seronegative arthopathy, arthropathy,Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy,enteropathic synovitis, chlamydia, yersinia and salmonella associatedarthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis,atopic allergy, autoimmune bullous disease, pemphigus vulgaris,pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmunehaemolytic anaemia, Coombs positive haemolytic anaemia, acquiredpernicious anaemia, juvenile pernicious anaemia, myalgicencephalitis/Royal Free Disease, chronic mucocutaneous candidiasis,giant cell arteritis, primary sclerosing hepatitis, cryptogenicautoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome,Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C,common varied immunodeficiency (common variable hypogammaglobulinaemia),dilated cardiomyopathy, female infertility, ovarian failure, prematureovarian failure, fibrotic lung disease, cryptogenic fibrosingalveolitis, post-inflammatory interstitial lung disease, interstitialpneumonitis, connective tissue disease associated interstitial lungdisease, mixed connective tissue disease associated lung disease,systemic sclerosis associated interstitial lung disease, rheumatoidarthritis associated interstitial lung disease, systemic lupuserythematosus associated lung disease, dermatomyositis/polymyositisassociated lung disease, Sjögren' s disease associated lung disease,ankylosing spondylitis associated lung disease, vasculitic diffuse lungdisease, haemosiderosis associated lung disease, drug-inducedinterstitial lung disease, fibrosis, radiation fibrosis, bronchiolitisobliterans, chronic eosinophilic pneumonia, lymphocytic infiltrativelung disease, postinfectious interstitial lung disease, gouty arthritis,autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmuneor lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibodyhepatitis), autoimmune mediated hypoglycaemia, type B insulin resistancewith acanthosis nigricans, hypoparathyroidism, acute immune diseaseassociated with organ transplantation, chronic immune disease associatedwith organ transplantation, osteoarthrosis, primary sclerosingcholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,autoimmune neutropaenia, renal disease NOS, glomerulonephritides,microscopic vasulitis of the kidneys, lyme disease, discoid lupuserythematosus, male infertility idiopathic or NOS, sperm autoimmunity,multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonaryhypertension secondary to connective tissue disease, Goodpasture'ssyndrome, pulmonary manifestation of polyarteritis nodosa, acuterheumatic fever, rheumatoid spondylitis, Still's disease, systemicsclerosis, Sjögren's syndrome, Takayasu's disease/arteritis, autoimmunethrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroiddisease, hyperthyroidism, goitrous autoimmune hypothyroidism(Hashimoto's disease), atrophic autoimmune hypothyroidism, primarymyxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liverdisease, chronic liver diseases, alcoholic cirrhosis, alcohol-inducedliver injury, choleosatatis, idiosyncratic liver disease, Drug-Inducedhepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group Bstreptococci (GB S) infection, mental disorders (e.g., depression andschizophrenia), Th2 Type and Th1 Type mediated diseases, acute andchronic pain (different forms of pain), and cancers such as lung,breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectalcancer and hematopoietic malignancies (leukemia and lymphoma). The humanantibodies, and antibody portions of the present application can be usedto treat humans suffering from autoimmune diseases, in particular thoseassociated with inflammation, including, rheumatoid spondylitis,allergy, autoimmune diabetes, autoimmune uveitis.

Non-limiting examples of autoimmune diseases include acquiredimmunodeficiency disease syndrome (AIDS), autoimmune lymphoproliferativesyndrome, hemolytic anemia, inflammatory diseases, and thrombocytopenia,acute or chronic immune disease associated with organ transplantation,Addison's disease, allergic diseases, alopecia, alopecia areata,atheromatous disease/arteriosclerosis, atherosclerosis, arthritis(including osteoarthritis, juvenile chronic arthritis, septic arthritis,Lyme arthritis, psoriatic arthritis and reactive arthritis), autoimmunebullous disease, abetalipoprotemia, acquired immunodeficiency-relateddiseases, acute immune disease associated with organ transplantation,acquired acrocyanosis, acute and chronic parasitic or infectiousprocesses, acute pancreatitis, acute renal failure, acute rheumaticfever, acute transverse myelitis, adenocarcinomas, aerial ectopic beats,adult (acute) respiratory distress syndrome, AIDS dementia complex,alcoholic cirrhosis, alcohol-induced liver injury, alcohol-inducedhepatitis, allergic conjunctivitis, allergic contact dermatitis,allergic rhinitis, allergy and asthma, allograft rejection,alpha-1-antitrypsin deficiency, Alzheimer's disease, amyotrophic lateralsclerosis, anemia, angina pectoris, ankylosing spondylitis associatedlung disease, anterior horn cell degeneration, antibody mediatedcytotoxicity, antiphospholipid syndrome, anti-receptor hypersensitivityreactions, aortic and peripheral aneurysms, aortic dissection, arterialhypertension, arteriosclerosis, arteriovenous fistula, arthropathy,asthenia, asthma, ataxia, atopic allergy, atrial fibrillation (sustainedor paroxysmal), atrial flutter, atrioventricular block, atrophicautoimmune hypothyroidism, autoimmune haemolytic anaemia, autoimmunehepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoidhepatitis), autoimmune mediated hypoglycaemia, autoimmune neutropaenia,autoimmune thrombocytopaenia, autoimmune thyroid disease, B celllymphoma, bone graft rejection, bone marrow transplant (BMT) rejection,bronchiolitis obliterans, bundle branch block, burns, cachexia, cardiacarrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy,cardiopulmonary bypass inflammation response, cartilage transplantrejection, cerebellar cortical degenerations, cerebellar disorders,chaotic or multifocal atrial tachycardia, chemotherapy associateddisorders, chlamydia, choleosatatis, chronic alcoholism, chronic activehepatitis, chronic fatigue syndrome, chronic immune disease associatedwith organ transplantation, chronic eosinophilic pneumonia, chronicinflammatory pathologies, chronic mucocutaneous candidiasis, chronicobstructive pulmonary disease (COPD), chronic salicylate intoxication,colorectal common varied immunodeficiency (common variablehypogammaglobulinaemia), conjunctivitis, connective tissue diseaseassociated interstitial lung disease, contact dermatitis, Coombspositive haemolytic anaemia, cor pulmonale, Creutzfeldt-Jakob disease,cryptogenic autoimmune hepatitis, cryptogenic fibrosing alveolitis,culture negative sepsis, cystic fibrosis, cytokine therapy associateddisorders, Crohn's disease, dementia pugilistica, demyelinatingdiseases, dengue hemorrhagic fever, dermatitis, dermatitis scleroderma,dermatologic conditions, dermatomyositis/polymyositis associated lungdisease, diabetes, diabetic arteriosclerotic disease, diabetes mellitus,Diffuse Lewy body disease, dilated cardiomyopathy, dilated congestivecardiomyopathy, discoid lupus erythematosus, disorders of the basalganglia, disseminated intravascular coagulation, Down's Syndrome inmiddle age, drug-induced interstitial lung disease, drug-inducedhepatitis, drug-induced movement disorders induced by drugs which blockCNS dopamine, receptors, drug sensitivity, eczema, encephalomyelitis,endocarditis, endocrinopathy, enteropathic synovitis, epiglottitis,Epstein-Barr virus infection, erythromelalgia, extrapyramidal andcerebellar disorders, familial hematophagocytic lymphohistiocytosis,fetal thymus implant rejection, Friedreich's ataxia, functionalperipheral arterial disorders, female infertility, fibrosis, fibroticlung disease, fungal sepsis, gas gangrene, gastric ulcer, giant cellarteritis, glomerular nephritis, glomerulonephritides, Goodpasture'ssyndrome, goitrous autoimmune hypothyroidism (Hashimoto's disease),gouty arthritis, graft rejection of any organ or tissue, graft versushost disease, gram negative sepsis, gram positive sepsis, granulomas dueto intracellular organisms, group B streptococci (GBS) infection,Grave's disease, haemosiderosis associated lung disease, hairy cellleukemia, hairy cell leukemia, Hallerrorden-Spatz disease, Hashimoto'sthyroiditis, hay fever, heart transplant rejection, hemachromatosis,hematopoietic malignancies (leukemia and lymphoma), hemolytic anemia,hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura,hemorrhage, Henoch-Schoenlein purpurea, Hepatitis A, Hepatitis B,Hepatitis C, HIV infection/HIV neuropathy, Hodgkin's disease,hypoparathyroidism, Huntington's chorea, hyperkinetic movementdisorders, hypersensitivity reactions, hypersensitivity pneumonitis,hyperthyroidism, hypokinetic movement disorders,hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison'sdisease, idiopathic leucopaenia, idiopathic pulmonary fibrosis,idiopathic thrombocytopaenia, idiosyncratic liver disease, infantilespinal muscular atrophy, infectious diseases, inflammation of the aorta,inflammatory bowel disease, insulin dependent diabetes mellitus,interstitial pneumonitis, iridocyclitis/uveitis/optic neuritis,ischemia-reperfusion injury, ischemic stroke, juvenile perniciousanaemia, juvenile rheumatoid arthritis, juvenile spinal muscularatrophy, Kaposi's sarcoma, Kawasaki's disease, kidney transplantrejection, legionella, leishmaniasis, leprosy, lesions of thecorticospinal system, linear IgA disease, lipidema, liver transplantrejection, Lyme disease, lymphederma, lymphocytic infiltrative lungdisease, malaria, male infertility idiopathic or NOS, malignanthistiocytosis, malignant melanoma, meningitis, meningococcemia,microscopic vasculitis of the kidneys, migraine headache, mitochondrialmultisystem disorder, mixed connective tissue disease, mixed connectivetissue disease associated lung disease, monoclonal gammopathy, multiplemyeloma, multiple systems degenerations (Mencel Dejerine-ThomasShi-Drager and Machado-Joseph), myalgic encephalitis/Royal Free Disease,myasthenia gravis, microscopic vasculitis of the kidneys, Mycobacteriumavium intracellulare, Mycobacterium tuberculosis, myelodyplasticsyndrome, myocardial infarction, myocardial ischemic disorders,nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis,nephrosis, nephrotic syndrome, neurodegenerative diseases, neurogenic Imuscular atrophies, neutropenic fever, Non-alcoholic Steatohepatitis,occlusion of the abdominal aorta and its branches, occlusive arterialdisorders, organ transplant rejection, orchitis/epidydimitis,orchitis/vasectomy reversal procedures, organomegaly, osteoarthrosis,osteoporosis, ovarian failure, pancreas transplant rejection, parasiticdiseases, parathyroid transplant rejection, Parkinson's disease, pelvicinflammatory disease, pemphigus vulgaris, pemphigus foliaceus,pemphigoid, perennial rhinitis, pericardial disease, peripheralatherlosclerotic disease, peripheral vascular disorders, peritonitis,pernicious anemia, phacogenic uveitis, Pneumocystis carinii pneumonia,pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy,monoclonal gammopathy, and skin changes syndrome), post perfusionsyndrome, post pump syndrome, post-MI cardiotomy syndrome,postinfectious interstitial lung disease, premature ovarian failure,primary biliary cirrhosis, primary sclerosing hepatitis, primarymyxoedema, primary pulmonary hypertension, primary sclerosingcholangitis, primary vasculitis, Progressive supranucleo Palsy,psoriasis, psoriasis type 1, psoriasis type 2, psoriatic arthropathy,pulmonary hypertension secondary to connective tissue disease, pulmonarymanifestation of polyarteritis nodosa, post-inflammatory interstitiallung disease, radiation fibrosis, radiation therapy, Raynaud'sphenomenon and disease, Raynoud's disease, Refsum's disease, regularnarrow QRS tachycardia, Reiter's disease, renal disease NOS,renovascular hypertension, reperfusion injury, restrictivecardiomyopathy, rheumatoid arthritis associated interstitial lungdisease, rheumatoid spondylitis, sarcoidosis, Schmidt's syndrome,scleroderma, senile chorea, Senile Dementia of Lewy body type, sepsissyndrome, septic shock, seronegative arthropathies, shock, sickle cellanemia, Sjögren's disease associated lung disease, Sjörgren's syndrome,skin allograft rejection, skin changes syndrome, small bowel transplantrejection, sperm autoimmunity, multiple sclerosis (all subtypes), spinalataxia, spinocerebellar degenerations, spondyloarthropathy,spondyloarthopathy, sporadic, polyglandular deficiency type I sporadic,polyglandular deficiency type II, Still's disease, streptococcalmyositis, stroke, structural lesions of the cerebellum, Subacutesclerosing panencephalitis, sympathetic ophthalmia, Syncope, syphilis ofthe cardiovascular system, systemic anaphylaxis, systemic inflammatoryresponse syndrome, systemic onset juvenile rheumatoid arthritis,systemic lupus erythematosus, systemic lupus erythematosus-associatedlung disease, systemic sclerosis, systemic sclerosis-associatedinterstitial lung disease, T-cell or FAB ALL, Takayasu'sdisease/arteritis, Telangiectasia, Th2 Type and Th1 Type mediateddiseases, thromboangitis obliterans, thrombocytopenia, thyroiditis,toxicity, toxic shock syndrome, transplants, trauma/hemorrhage, type-2autoimmune hepatitis (anti-LKM antibody hepatitis), type B insulinresistance with acanthosis nigricans, type III hypersensitivityreactions, type IV hypersensitivity, ulcerative colitic arthropathy,ulcerative colitis, unstable angina, uremia, urosepsis, urticaria,uveitis, valvular heart diseases, varicose veins, vasculitis, vasculiticdiffuse lung disease, venous diseases, venous thrombosis, ventricularfibrillation, vitiligo acute liver disease, viral and fungal infections,vital encephalitis/aseptic meningitis, vital-associated hemaphagocyticsyndrome, Wegener's granulomatosis, Wernicke-Korsakoff syndrome,Wilson's disease, xenograft rejection of any organ or tissue, yersiniaand salmonella-associated arthropathy and the like.

Nitroxide Antioxidant

Non-limiting examples of the nitroxide antioxidant include2-ethyl-2,5,5-trimethyl-3-oxazolidine-1-oxyl (OXANO),2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO),4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), 4-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (Tempamine), 3-Aminomethyl-PROXYL, 3-Cyano-PROXYL, 3-Carbamoyl-PROXYL, 3-Carboxy-PROXYL,and 4-Oxo-TEMPO. TEMPO can also be substituted, typically in the 4position, for example, 4-amino, 4-(2-bromoacetamido),4-(ethoxyfluorophosphonyloxy), 4-hydroxy, 4-(2-iodoacetamido),4-isothiocyanato, 4-maleimido, 4-(4-nitrobenzoyloxyl), 4-phosphonooxy,and the like.

The use of other nitroxide compounds is also contemplated. According tocertain embodiments the nitroxide compound can be selected from thefollowing formulas:

wherein X is selected from O— and OH, and R is selected from COOH, CONH,CN, and CH2NH2;

wherein X is selected from O— and OH, and R1 is selected from CH3 andspirocyclohexyl, and R2 is selected from C2H5 and spirocyclohexyl;

wherein X is selected from O— and OH and R is selected from CONH; and

wherein X is selected from O— and OH and R is selected from H, OH, andNH2.

Suitable nitroxide compounds can also be found in Proctor, U.S. Pat. No.5,352,442, and Mitchell et al., U.S. Pat. No. 5,462,946, both of whichare hereby incorporated by reference in their entireties.

In some embodiments, the nitroxide antioxidant includes or is associatedwith (e.g., binds to or is conjugated with) a bioeffector molecule. Forexample, the bioeffector molecule is a targeting subunit bound to thenitroxide antioxidant, such as a mitochondrial targeting subunit. Atargeting subunit can direct activity of the nitroxide antioxidant to apredetermined location within or on the cell. Non-limiting examples ofmitochondrial targeting bioeffector molecules includestriphenylphosphine (TPP), gramicidin, and any functional groupeffectively charged to be attracted to the polarized mitochondria.

In some embodiments, the nitroxide antioxidant is structurally cyclichaving a ring structure including a nitroxide molecule incorporatedtherein. In some embodiments, the nitroxide antioxidant is characterizedas the nitroxide molecule functioning as the catalytic center.

Dosage

In some embodiments, the nitroxide antioxidant, non-toxic salts thereof,acid addition salts thereof or hydrates thereof may be administeredsystemically or locally, usually by oral or parenteral administration.The doses to be administered can be determined depending upon, forexample, age, body weight, symptom, the desired therapeutic effect, theroute of administration, and the duration of the treatment. In the humanadult, the dose per person at a time can be generally from about 0.01 toabout 1000 mg, by oral administration, up to several times per day.Specific examples of particular amounts contemplated via oraladministration include about 0.02, 0.03, 0.04, 0.05, 0.10, 0.15, 0.20,0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80,0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260,265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330,335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400,405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470,475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540,545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610,615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680,685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750,755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 820, 825,830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895,900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965,970, 975, 980, 985, 990, 995, 1000 or more mg. The dose per person at atime can be generally from about 0.01 to about 300 mg/kg via parenteraladministration (preferably intravenous administration), from one to fouror more times per day. Specific examples of particular amountscontemplated include about 0.02, 0.03, 0.04, 0.05, 0.10, 0.15, 0.20,0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80,0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260,265, 270, 275, 280, 285, 290, 295, 300 or more mg/kg. Continuousintravenous administration can also contemplated for from 1 to 24 hoursper day to achieve a target concentration from about 0.01 mg/L to about100 mg/L. Non-limiting examples of particular amounts contemplated viathis route include about 0.02, 0.03, 0.04, 0.05, 0.10, 0.15, 0.20, 0.25,0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85,0.90, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more mg/L. The dose to beused does can depend upon various conditions, and there may be caseswherein doses lower than or greater than the ranges specified above areused.

Compositions

The nitroxide antioxidant can be administered in the form of, forexample, solid compositions, liquid compositions or other compositionsfor oral administration, injections, liniments or suppositories forparenteral administration.

Solid compositions for oral administration include compressed tablets,pills, capsules, dispersible powders and granules. Capsules include hardcapsules and soft capsules. In such solid compositions, Tempol may beadmixed with an excipient (e.g. lactose, mannitol, glucose,microcrystalline cellulose, starch), combining agents (hydroxypropylcellulose, polyvinyl pyrrolidone or magnesium metasilicate aluminate),disintegrating agents (e.g. cellulose calcium glycolate), lubricatingagents (e.g. magnesium stearate), stabilizing agents, agents to assistdissolution (e.g. glutamic acid or aspartic acid), or the like. Theagents may, if desired, be coated with coating agents (e.g. sugar,gelatin, hydroxypropyl cellulose or hydroxypropylmethyl cellulosephthalate), or be coated with two or more films. Further, coating mayinclude containment within capsules of absorbable materials such asgelatin.

Liquid compositions for oral administration include pharmaceuticallyacceptable solutions, suspensions, emulsions, syrups and elixirs. Insuch compositions, the nitroxide antioxidant is dissolved, suspended oremulsified in a commonly used diluent (e.g. purified water, ethanol ormixture thereof). Furthermore, such liquid compositions may alsocomprise wetting agents or suspending agents, emulsifying agents,sweetening agents, flavoring agents, perfuming agents, preservingagents, buffer agents, or the like.

Injections for parenteral administration include solutions, suspensions,emulsions and solids which are dissolved or suspended. For injections,the nitroxide antioxidant can be dissolved, suspended and emulsified ina solvent. The solvents include, for example, distilled water forinjection, physiological salt solution, vegetable oil, propylene glycol,polyethylene glycol, alcohol such as ethanol, or a mixture thereof.Moreover the injections can also include stabilizing agents, agents toassist dissolution (e.g. glutamic acid, aspartic acid or POLYS ORBATE80™), suspending agents, emulsifying agents, soothing agents, bufferagents, preserving agents, etc. They can be sterilized in the finalprocess or manufactured and prepared by sterile procedure. They can alsobe manufactured in the form of sterile solid compositions, such as afreeze-dried composition, and they may be sterilized or dissolvedimmediately before use in sterile distilled water for injection or someother solvent.

Other compositions for parenteral administration include liquids forexternal use, and ointment, endermic liniments, inhale, spray,suppositories for rectal administration and pessaries for vaginaladministration which comprise the nixtroxide antioxidant and areadministered by methods known in the art.

Spray compositions can comprise additional substances other thandiluents: e.g. stabilizing agents (e.g. sodium sulfite hydride),isotonic buffers (e.g. sodium chloride, sodium citrate or citric acid).A small aerosol particle size useful for effective distribution of themedicament can be obtained by employing self-propelling compositionscontaining the drugs in micronized form dispersed in a propellantcomposition. Effective dispersion of the finely divided drug particlescan be accomplished with the use of very small quantities of asuspending agent, present as a coating on the micronized drug particles.Evaporation of the propellant from the aerosol particles after sprayingfrom the aerosol container leaves finely divided drug particles coatedwith a fine film of the suspending agent. In the micronized form, theaverage particle size can be less than about 5 microns. The propellantcomposition may employ, as the suspending agent, a fatty alcohol such asoleyl alcohol. The minimum quantity of suspending agent can beapproximately 0.1 to 0.2 percent by weight of the total composition. Theamount of suspending agent can be less than about 4 percent by weight ofthe total composition to maintain an upper particle size limit of lessthan 10 microns or 5 microns. Propellants that may be employed includehydrofluoroalkane propellants and chlorofluorocarbon propellants. Drypowder inhalation may also be employed.

EXAMPLES

Some aspects of the embodiments discussed above are disclosed in furtherdetail in the following examples, which are not in any way intended tolimit the scope of the present disclosure.

In order to facilitate understanding, the specific embodiments areprovided to help interpret the technical proposal, that is, theseembodiments are only for illustrative purposes, but not in any way tolimit the scope of the invention. Unless otherwise specified,embodiments do not indicate the specific conditions, are in accordancewith the conventional conditions or the manufacturer's recommendedconditions.

Example 1. Effects of Tempol on Expression of Genes Associated WithProteasome Activity

To assess the effects of Tempol on gene expression, Tempol wasadministered to experimental mice at a dose of 5 mg/g of food from 14months to 31 months after birth. Mice receiving the same food withoutthe addition of Tempol were used as a negative control. At the age of 31months, the experimental animals were sacrificed and the hearts weresurgically removed. The expression of a broad spectrum of genes in thecardiac tissue was assessed using chip-based microarray technology. Suchchips are well known in the art and are widely used to assess geneexpression. The experimental results showed that five genes associatedwith proteasome activity, Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4,Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, andPsmc4, exhibited statistically significant decrease in expression. Thisresult is shown in Table 1.

TABLE 1 Genes Associated With Proteasome Activity Exhibiting DecreasedExpression In White Adipose Tissue After Tempol Administration Tempol-Control treated Fold Symbol Gene title mice mice change P-value Psma6Alpha type subunit 6 3782 3467 −1.09 0.04 Psmd3 Non-ATPase 26s 1146 1017−1.12 0.03 subunit 3 Psmb4 Beta type subunit 4 3622 3614 −1.15 0.02Psmb5 Best type subunit 5 2018 1754 −1.15 0.01 Psmc6 ATPase 26s subunit6 1686 1449 −1.16 0.03 Psme4 Activator subunit 4 1257 1086 −1.16 0.03Psmd9 Non-ATPase 26s 364 310 −1.18 0.01 subunit 9 Psmd12 Non-ATPase 26s1326 1098 −1.20 0.00 subunit 12 Psmd14 Non-ATPase 26s 3294 2745 −1.200.00 subunit 14 Psmd4 Non-ATPase 26s 3133 2610 −1.20 0.04 subunit 4Psma2 Alpha type subunit 2 3744 3123 −1.20 0.00 Psmc2 ATPase 26s subunit2 2689 2176 −1.23 0.01 Psmc3 ATPase 26s subunit 3 2235 1810 −1.23 0.00Psmd13 Non-ATPase 26s 1294 1047 −1.23 0.01 subunit 13 Psma7 Alpha typesubunit 7 1864 1513 −1.23 0.00 Psmc4 ATPase 26s subunit 4 1673 1101−1.52 0.02

Example 2. Treating Age-Related Increase in Gene Expression

A 70-kilogram human subject over the age of 65 is identified as having,or known to have, or suspected of having increased expression level ofPsma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4,Psma2, Psmc2, Psmc3, Psmd13, Psma7, or Psmc4. The human subject isadministered a dose of 1500 mg of Tempol (or another nitroxideantioxidant) per day for 180 days. This may be administered in a singledose, or may be administered as a number of smaller doses over a 24-hourperiod: for example, three 500-mg doses at eight-hour intervals.Following treatment, the serum level of Psma6, Psmd3, Psmb4, Psmb5,Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13,Psma7, or Psmc4, is decreased.

Example 3. Treating a Human Subject with Increased Gene Expression

A 70-kilogram human subject is identified as having, or known to have,or suspected of having increased expression level of Psma6, Psmd3,Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2,Psmc3, Psmd13, Psma7, or Psmc4. The human subject is administered a doseof 1500 mg of Tempol (or another nitroxide antioxidant) per day for 180days. This may be administered in a single dose, or may be administeredas a number of smaller doses over a 24-hour period: for example, three500-mg doses at eight-hour intervals. Following treatment, the serumlevel of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12,Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, or Psmc4, isdecreased.

Example 4. Treating a Human Subject with an Age-Related Disease

A 70-kilogram human subject over the age of 65 and having acardiovascular disease is identified for increased expression level ofPsma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4,Psma2, Psmc2, Psmc3, Psmd13, Psma7, or Psmc4. Or a 70-kilogram humansubject over the age of 65 is known to have a cardiovascular diseaseand/or increased expression level of Psma6, Psmd3, Psmb4, Psmb5, Psmc6,Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7,or Psmc4. The human subject is administered a dose of 1500 mg of Tempol(or another nitroxide antioxidant) per day for 180 days. This may beadministered in a single dose, or may be administered as a number ofsmaller doses over a 24-hour period: for example, three 500-mg doses ateight-hour intervals. Following treatment, the serum level of Psma6,Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2,Psmc2, Psmc3, Psmd13, Psma7, or Psmc4, is decreased.

Example 5. Treating a Human Subject at Risk of Developing Cancer

A 70-kilogram human subject at risk of developing colorectal cancer isidentified for increased expression level of Psma6, Psmd3, Psmb4, Psmb5,Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13,Psma7, or Psmc4. Or a 70-kilogram human subject is known to be at riskof developing colorectal cancer and/or have increased expression levelof Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14,Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, or Psmc4. The human subjectis administered a dose of 1500 mg of Tempol (or another nitroxideantioxidant) per day for 180 days. This may be administered in a singledose, or may be administered as a number of smaller doses over a 24-hourperiod: for example, three 500-mg doses at eight-hour intervals.Following treatment, the serum level of Psma6, Psmd3, Psmb4, Psmb5,Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13,Psma7, or Psmc4, is decreased.

Example 6. Treating a Human Subject at Risk of Developing an AutoimmuneDisease

A 70-kilogram human subject at risk of developing an autoimmune disease(e.g., rheumatoid arthritis) is identified for increased expressionlevel of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12,Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, or Psmc4. Or a70-kilogram human subject is known to be at risk of developing anautoimmune disease and/or have increased expression level of Psma6,Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2,Psmc2, Psmc3, Psmd13, Psma7, or Psmc4. The human subject is administereda dose of 1500 mg of Tempol (or another nitroxide antioxidant) per dayfor 180 days. This may be administered in a single dose, or may beadministered as a number of smaller doses over a 24-hour period: forexample, three 500-mg doses at eight-hour intervals. Followingtreatment, the serum level of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4,Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, orPsmc4, is decreased.

Example 7. Treating a Human Subject at Risk of Developing a ConditionDue to Aging

A 70-kilogram human subject of 45 years old at risk of developing acondition due to aging is identified. Or a 70-kilogram human subject of45 years old is known to be at risk of developing a condition. The humansubject is administered a dose of 1500 mg of Tempol (or anothernitroxide antioxidant) per day for 180 days. This may be administered ina single dose, or may be administered as a number of smaller doses overa 24-hour period: for example, three 500-mg doses at eight-hourintervals. Following treatment, the serum level of Psma6, Psmd3, Psmb4,Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3,Psmd13, Psma7, or Psmc4, is decreased.

Example 8. Treating a Human Subject at Risk of Developing aNeruodegenerative Disease

A 70-kilogram human subject at risk of developing a neurodegenerativedisease (e.g., Parkinson's Disease) is identified for increasedexpression level of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9,Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, or Psmc4. Ora 70-kilogram human subject is known to be at risk of developing aneurodegenerative disease and/or have increased expression level ofPsma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4,Psma2, Psmc2, Psmc3, Psmd13, Psma7, or Psmc4. The human subject isadministered a dose of 1500 mg of Tempol (or another nitroxideantioxidant) per day for 180 days. This may be administered in a singledose, or may be administered as a number of smaller doses over a 24-hourperiod: for example, three 500-mg doses at eight-hour intervals.Following treatment, the serum level of Psma6, Psmd3, Psmb4, Psmb5,Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13,Psma7, or Psmc4, is decreased.

Example 9. Treating a Human Subject Having an Infection

A 70-kilogram human subject having an infection (e.g., a bacterial,fungal, or viral infection) is identified for increased expression levelof Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14,Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, or Psmc4. Or a 70-kilogramhuman subject is known to have an infectione and/or have increasedexpression level of Psma6, Psmd3, Psmb4, Psmb5, Psmc6, Psme4, Psmd9,Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3, Psmd13, Psma7, or Psmc4. Thehuman subject is administered a dose of 1500 mg of Tempol (or anothernitroxide antioxidant) per day for 180 days. This may be administered ina single dose, or may be administered as a number of smaller doses overa 24-hour period: for example, three 500-mg doses at eight-hourintervals. Following treatment, the serum level of Psma6, Psmd3, Psmb4,Psmb5, Psmc6, Psme4, Psmd9, Psmd12, Psmd14, Psmd4, Psma2, Psmc2, Psmc3,Psmd13, Psma7, or Psmc4, is decreased.

In at least some of the previously described embodiments, one or moreelements used in an embodiment can interchangeably be used in anotherembodiment unless such a replacement is not technically feasible. Itwill be appreciated by those skilled in the art that various otheromissions, additions and modifications may be made to the methods andstructures described above without departing from the scope of theclaimed subject matter. All such modifications and changes are intendedto fall within the scope of the subject matter, as defined by theappended claims.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

What is claimed is:
 1. A method of decreasing expression level of a geneencoding a protein of a proteasome complex in a subject suffering from aviral infection, the method comprising: identifying the subject ashaving an increased expression level of a gene encoding a protein of aproteasome complex, the increased expression level associated with viralinfection, wherein the gene selected from the group consisting of Psma6,Psmd3, Psmb4 , Psmb5, Psmc6, Psme4,Psmd9, Psmd12, Psmd14, Psmd4, Psma2,Psmc2, Psmc3, Psmd13, Psma7, and Psmc4; and administering an effectiveamount of 4-hydroxy-2,2,6,6-tetramethylpiperidine- 1-oxyl (TEMPOL) tothe subject, whereby the expression level of the gene is decreased. 2.The method of claim 1, wherein the gene is Psma6.
 3. The method of claim1, wherein the gene is Psmd3.
 4. The method of claim 1, wherein the geneis Psmb4.
 5. The method of claim 1, wherein the gene is Psmb5.
 6. Themethod of claim 1, wherein the gene is Psmc6.
 7. The method of claim 1,wherein the gene is Psme4.
 8. The method of claim 1, wherein the gene isPsmd9.
 9. The method of claim 1, wherein the gene is Psmd12.
 10. Themethod of claim 1, wherein the gene is Psmd14.
 11. The method of claim1, wherein the gene is Psmd4.
 12. The method of claim 1, wherein thegene is Psma2.
 13. The method of claim 1, wherein the gene is Psmc2. 14.The method of claim 1, wherein the gene is Psmc3.
 15. The method ofclaim 1, wherein the gene is Psmd13.
 16. The method of claim 1, whereinthe gene is Psma7.
 17. The method of claim 1, wherein the gene is Psmc4.